Proteins binding nkg2d, cd16 and baff-r

Abstract

Multispecific binding proteins that bind NKG2D receptor, CD 16, and B cell-activating factor receptor (BAFF-R) are described, as well as pharmaceutical compositions and therapeutic methods of the multispecific binding proteins useful for the treatment of cancer and autoimmune inflammatory diseases.

Description

PROTEINS BINDING NKG2D, CD16 AND BAFF-RCROSS REFERENCE

[0001] This application claims the benefit of US Provisional Application No. 63 / 250,160, filed September 29, 2021, the full disclosure of which is hereby incorporated by reference herein in its entirety.SEQUENCE LISTING

[0002] This application contains a computer readable Sequence Listing which has been submitted in XML file format via Patent Center, the entire content of which is incorporated by reference herein in its entirety. The Sequence Listing XML file submitted via Patent Center is entitled “14247-700-228_seqlist.xml,” was created on September 16, 2022, and is 305,046 bytes in size.FIELD OF THE INVENTION

[0003] The present application relates to multispecific binding proteins that bind to NKG2D, CD 16, and B cell-activating factor receptor (BAFF-R) on a cell, pharmaceutical compositions comprising such proteins, and therapeutic methods using such proteins and pharmaceutical compositions, including for the treatment of cancer.BACKGROUND

[0004] Despite substantial research efforts, cancer continues to be a significant clinical and financial burden in countries across the globe. According to the World Health Organization (WHO), it is the second leading cause of death. Surgery, radiation therapy, chemotherapy, biological therapy, immunotherapy, hormone therapy, stem-cell transplantation, and precision medicine are among the existing treatment modalities. Despite extensive research in these areas, a highly effective, curative solution, particularly for the most aggressive cancers, has yet to be identified. Furthermore, many of the existing anti-cancer treatment modalities have substantial adverse side effects.

[0005] Cancer immunotherapies are desirable because they are highly specific and can facilitate destruction of cancer cells using the patient’s own immune system. Fusion proteinssuch as bi-specific T-cell engagers are cancer immunotherapies described in the literature that bind to tumor cells and T-cells to facilitate destruction of tumor cells.

[0006] Natural killer (NK) cells are a component of the innate immune system and make up approximately 15% of circulating lymphocytes. NK cells infiltrate virtually all tissues and were originally characterized by their ability to kill tumor cells effectively without the need for prior sensitization. Activated NK cells kill target cells by means similar to cytotoxic T cells - / .< ., via cytolytic granules that contain perforin and granzymes as well as via death receptor pathways. Activated NK cells also secrete inflammatory cytokines such as IFN-y and chemokines that promote the recruitment of other leukocytes to the target tissue.

[0007] NK cells respond to signals through a variety of activating and inhibitory receptors on their surface. For example, when NK cells encounter healthy self-cells, their activity is inhibited through activation of the killer-cell immunoglobulin-like receptors (KIRs). Alternatively, when NK cells encounter foreign cells or cancer cells, they are activated via their activating receptors (e.g., NKG2D, NCRs, DNAM1). NK cells are also activated by the constant region of some immunoglobulins through CD 16 receptors on their surface. The overall sensitivity of NK cells to activation depends on the sum of stimulatory and inhibitory signals. NKG2D is a type-II transmembrane protein that is expressed by essentially all natural killer cells where NKG2D serves as an activating receptor. NKG2D is also be found on T cells where it acts as a costimulatory receptor. The ability to modulate NK cell function via NKG2D is useful in various therapeutic contexts including malignancy.

[0008] BAFF-R, also called BAFF receptor, TNF receptor superfamily member 13C (TNFRSF13C), CD268, or BR3, is a type III transmembrane protein of the TNF receptor superfamily. BAFF-R is expressed at the late transitional (T2) B-cell stage and on all mature B cells, is downregulated on germinal center B cells, is re-expressed on memory cells, and is absent on plasma cells (Davidson (2012) Curr. Rheumatol. Rep., 14(4): 295-302). BAFF-R is a receptor for B cell-activating factor (BAFF), a B cell survival factor. BAFF can engage three receptors: BAFF-R, transmembrane activator and CAML interactor (TACI), and B-cell maturation antigen (BCMA). Among these three receptors, BAFF-R is the principal receptor involved in the development of follicular and marginal zone splenic B cells (Schiemann et al. (2001) Science, 293: 2111-14).

[0009] The BAFF / BAFF-R signaling axis may play a role in B cell hyperplasia. Increased expression of BAFF-R, as well as elevated serum levels of BAFF, has been observed in nonHodgkin lymphoma (NHL) patients (Shen et al. (2016) Adv. Clin. Exp. Med., 25(5):837-44). Certain single nucleotide polymorphisms (SNPs) in BAFF-R are associated with increased risk of chronic lymphocytic leukemia (CLL) (Jesek et al. (2016) Tumour Biol., 37(10): 13617-26). The BAFF / BAFF-R axis is also implicated in autoimmune inflammatory diseases (Mackay et al. (1999) J. Exp. Med., 190: 1697-1710). Some systemic lupus erythematosus (SLE) patients have increased levels of BAFF in serum (Cheema et al. (2001) Arthritis Rheum., 44: 1313-19), and BAFF-R is consistently occupied on blood B cells in SLE (Carter et al. (2005) Arthritis Rheum., 52:3943-54). Given the observation that autoreactive B cells have a greater dependency on BAFF for their survival as compared with protective B cells (Lesley et al. (2004) Immunity, 20:441-53), it has been proposed that abnormally high levels of BAFF may contribute to the pathogenesis of autoimmune diseases by enhancing the survival of autoreactive B cells.

[0010] Therefore, there remains a need in the field for new and useful proteins that bind BAFF-R for use in treatment of cancer and autoimmune inflammatory diseases.SUMMARY

[0011] The present application provides multispecific binding proteins that bind to the NKG2D receptor and CD 16 receptor on natural killer cells, and BAFF-R. Such proteins can engage more than one kind of NK-activating receptor, and may block the binding of natural ligands to NKG2D. In certain embodiments, the proteins can agonize NK cells in humans. In some embodiments, the proteins can agonize NK cells in humans and in other species such as rodents and cynomolgus monkeys. Formulations containing any one of the proteins disclosed herein; cells containing one or more nucleic acids expressing the proteins, and methods of enhancing tumor cell death using the proteins are also provided.

[0012] Accordingly, in one aspect, the present application provides a protein comprising:(a) a first antigen-binding site that binds NKG2D;(b) a second antigen-binding site that binds B cell-activating factor receptor (BAFF-R); and(c) an antibody Fc domain or a portion thereof sufficient to bind CD 16, or a third antigen-binding site that binds CD 16.

[0013] In some embodiments of a protein disclosed herein, the first antigen-binding site that binds NKG2D is a Fab fragment, and the second antigen-binding site that binds BAFF-R is an scFv. In some embodiments, the first antigen-binding site that binds NKG2D is an scFv, and the second antigen-binding site that binds BAFF-R is a Fab fragment.

[0014] In some embodiments of a protein disclosed herein, the protein further comprises an additional antigen-binding site that binds BAFF-R. In certain embodiments, the first antigenbinding site that binds NKG2D is an scFv, and the second and the additional antigen-binding sites that bind BAFF-R are each a Fab fragment. In certain embodiments, the first antigenbinding site that binds NKG2D is an scFv, and the second and the additional antigen-binding sites that bind BAFF-R are each an scFv. In certain embodiments, the amino acid sequences of the second and the additional antigen-binding sites are identical. In certain embodiments, the amino acid sequences of the second and the additional antigen-binding sites are different.

[0015] In some embodiments of a protein disclosed herein, the scFv that binds NKG2D is linked to an antibody constant domain or a portion thereof sufficient to bind CD 16, via a hinge comprising Ala-Ser or Gly-Ser, wherein the scFv comprises a heavy chain variable domain and a light chain variable domain. In certain embodiments, each scFv that binds BAFF-R is linked to an antibody constant domain or a portion thereof sufficient to bind CD 16, via a hinge comprising Ala-Ser or Gly-Ser, wherein the scFv comprises a heavy chain variable domain and a light chain variable domain. In certain embodiments, the hinge further comprises an amino acid sequence Thr-Lys-Gly.

[0016] In some embodiments of a protein disclosed herein, within the scFv that binds NKG2D, the heavy chain variable domain of the scFv forms a disulfide bridge with the light chain variable domain of the scFv. In some embodiments, within each scFv that binds BAFF-R, the heavy chain variable domain of the scFv forms a disulfide bridge with the light chain variable domain of the scFv. In some embodiments, the disulfide bridge is formed between C44 of the heavy chain variable domain and Cl 00 of the light chain variable domain, numbered under the Kabat numbering scheme. In some embodiments, within the scFv that binds NKG2D, the heavy chain variable domain is linked to the light chain variable domain via a flexible linker. In some embodiments, within each scFv that binds BAFF-R, the heavy chain variable domain is linked to the light chain variable domain via a flexible linker. In certain embodiments, the flexible linkercomprises (GrS In certain embodiments, within the scFv that binds NKG2D, the heavy chain variable domain is positioned at the C-terminus of the light chain variable domain. In certain embodiments, within each scFv that binds BAFF-R, the heavy chain variable domain is positioned at the C-terminus of the light chain variable domain. In certain embodiments, within the scFv that binds NKG2D, the heavy chain variable domain is positioned at the N-terminus of the light chain variable domain. In certain embodiments, within each scFv that binds BAFF-R, the heavy chain variable domain is positioned at the N-terminus of the light chain variable domain. In certain embodiments, the Fab fragment that binds NKG2D is not positioned between an antigen-binding site and the Fc or the portion thereof. In certain embodiments, no Fab fragment that binds BAFF-R is positioned between an antigen-binding site and the Fc or the portion thereof.

[0017] In another aspect, provided herein is a protein comprising:(a) a first antigen-binding site comprising a Fab fragment that binds NKG2D;(b) a second antigen-binding site comprising a single-chain variable fragment (scFv) that binds B cell-activating factor receptor (BAFF-R); and(c) an Fc domain comprising a first antibody constant domain and a second antibody constant domain that form a heterodimer that binds CD 16, wherein the scFv is linked to the N-terminus of the first antibody constant domain via a hinge, and the Fab is linked to the N-terminus of the second antibody constant domain.

[0018] In some embodiments, the hinge comprises Gly-Ser.

[0019] In some embodiments of a protein disclosed herein, the first antigen-binding site binds human NKG2D. In some embodiments, the first antigen-binding site that binds NKG2D comprises a VH comprising complementarity-determining region 1 (CDR1), complementaritydetermining region 2 (CDR2), and complementarity-determining region 3 (CDR3) comprising the amino acid sequences of SEQ ID NOs: 81, 82, and 112, respectively; and a VL comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 86, 77, and 87, respectively. In some embodiments, the first antigen-binding site that binds NKG2D comprises a VH comprising CDR1, CDR2, and CDR3 sequences represented by the amino acid sequences of SEQ ID NOs: 81, 82, and 97, respectively; and a VL comprising CDR1, CDR2, and CDR3sequences represented by the amino acid sequences of SEQ ID NOs: 86, 77, and 87, respectively. In some embodiments, the first antigen-binding site that binds NKG2D comprises a VH comprising an amino acid sequence at least 90% identical to SEQ ID NO: 95 and a VL comprising an amino acid sequence at least 90% identical to SEQ ID NO:85. In certain embodiments, the first antigen-binding site that binds NKG2D comprises a VH comprising an amino acid sequence of SEQ ID NO:95 and a VL comprising an amino acid sequence of SEQ ID NO:85.

[0020] In some embodiments of a protein disclosed herein, the second antigen-binding site comprises a heavy chain variable domain comprising CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 260, 249, and 261, respectively; and a light chain variable domain comprising CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 217, 77, and 259, respectively.

[0021] In some embodiments of a protein disclosed herein, the second antigen-binding site comprises a heavy chain variable domain comprising CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 214, 233, and 248, respectively; and a light chain variable domain comprising CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 217, 77, and 249, respectively. In some embodiments, the second antigen-binding site comprises a heavy chain variable domain at least 90% identical to SEQ ID NO:250 and a light chain variable domain at least 90% identical to SEQ ID NO:251.

[0022] In some embodiments of a protein disclosed herein, the second antigen-binding site comprises a VH with a G44C substitution relative to SEQ ID NO:250, and a VL with a G100C substitution relative to SEQ ID NO:251. In some embodiments, the second antigen-binding site comprises a VH comprising the amino acid sequence of SEQ ID NO:252 and a VL comprising the amino acid sequence of SEQ ID NO:253, or a VH comprising the amino acid sequence of SEQ ID NO:250 and a VL comprising the amino acid sequence of SEQ ID NO:251. In some embodiments, the second antigen-binding site comprises a VH comprising the amino acid sequence of SEQ ID NO:252 and a VL comprising the amino acid sequence of SEQ ID NO:253. In some embodiments, the second antigen-binding site comprises a VH comprising the amino acid sequence of SEQ ID NO:250 and a VL comprising the amino acid sequence of SEQ ID NO:251.

[0023] In some embodiments of a protein disclosed herein, the second antigen-binding site comprises a single-chain fragment variable (scFv), and the scFv comprises a VH comprising the amino acid sequence of SEQ ID NO:252 and a VL comprising the amino acid sequence of SEQ ID NO:253. In some embodiments, the second antigen-binding site comprises an scFv and the the scFv comprises an amino acid sequence at least 90% identical to a sequence selected from the group consisting of SEQ ID NOs: 254 and 255. In some embodiments, the second antigenbinding site comprises an scFv and the scFv comprises an amino acid sequence at least 90% identical to SEQ ID NO:254. In some embodiments, the second antigen-binding site comprises an scFv and the scFv comprises an amino acid sequence of SEQ ID NO:254.

[0024] In some embodiments of a protein disclosed herein, the protein comprises an amino acid sequence at least 90% identical to SEQ ID NO:270. In some embodiments, the protein comprises an amino acid sequence of SEQ ID NO:270. In some embodiments, the protein comprises an amino acid sequence at least 90% identical to SEQ ID NO:271. In some embodiments, the protein comprises an amino acid sequence of SEQ ID NO:271.

[0025] In some embodiments of a protein disclosed herein, the second antigen-binding site binds human BAFF-R with a dissociation constant (KD) smaller than or equal to 5 nM, as measured by surface plasmon resonance (SPR).

[0026] In some embodiments of a protein disclosed herein, the second antigen-binding site inhibits (e.g., blocks) binding of BAFF-R to BAFF (e.g., by at least 50%, at least 75%, at least 90%, at least 95% or at least 99% as measured in a competitive binding assay).

[0027] In another aspect, provided herein is a protein comprising:(a) a first antigen-binding site comprising a VH and a VL of an anti-NKG2D antibody, wherein the VH comprises the amino acid sequence of SEQ ID NO:95 and the VL comprises the amino acid sequence of SEQ ID NO:85;(b) a second antigen-binding site comprising a VH and a VL of an anti-BAFF-R antibody, wherein the VH comprises the amino acid sequence of SEQ ID NO:252 and the VL comprises the amino acid sequence of SEQ ID NO:253; and(c) an antibody Fc domain or a portion thereof sufficient to bind CD 16, or a third antigen-binding site that binds CD 16.

[0028] In another aspect, provided herein is a protein comprising:(a) a first antigen-binding site comprising a VH and a VL of an anti-NKG2D antibody, wherein the VH comprises the amino acid sequence of SEQ ID NO:95 and the VL comprises the amino acid sequence of SEQ ID NO:85;(b) a second antigen-binding site comprising the amino acid sequence of SEQ ID NO:254; and(c) an antibody Fc domain or a portion thereof sufficient to bind CD 16, or a third antigen-binding site that binds CD 16.

[0029] In some embodiments of a protein disclosed herein, the antibody Fc domain is a human IgGl antibody Fc domain. In some embodiments, the antibody Fc domain or the portion thereof comprises an amino acid sequence at least 90% identical to SEQ ID NO: 118. In certain embodiments, at least one polypeptide chain of the antibody Fc domain comprises one or more mutations, relative to SEQ ID NO: 118, at one or more positions selected from Q347, Y349, L351, S354, E356, E357, K360, Q362, S364, T366, L368, K370, N390, K392, T394, D399, S400, D401, F405, Y407, K409, T411, and K439, numbered according to the EU numbering system. In certain embodiments, at least one polypeptide chain of the antibody Fc domain comprises one or more mutations, relative to SEQ ID NO:118, selected from Q347E, Q347R, Y349S, Y349K, Y349T, Y349D, Y349E, Y349C, L351K, L351D, L351Y, S354C, E356K, E357Q, E357L, E357W, K360E, K360W, Q362E, S364K, S364E, S364H, S364D, T366V, T366I, T366L, T366M, T366K, T366W, T366S, L368E, L368A, L368D, K370S, N390D, N390E, K392L, K392M, K392V, K392F, K392D, K392E, T394F, D399R, D399K, D399V, S400K, S400R, D401K, F405A, F405T, F405L, Y407A, Y407I, Y407V, K409F, K409W, K409D, K409R, T41 ID, T41 IE, K439D, and K439E, numbered according to the EU numbering system. In certain embodiments, one polypeptide chain of the antibody heavy chain constant region comprises one or more mutations, relative to SEQ ID NO: 118, at one or more positions selected from Q347, Y349, L351, S354, E356, E357, K360, Q362, S364, T366, L368, K370, K392, T394, D399, S400, D401, F405, Y407, K409, T411 and K439; and the other polypeptide chain of the antibody heavy chain constant region comprises one or more mutations, relative to SEQ ID NO: 118, at one or more positions selected from Q347, Y349, L351, S354, E356, E357, S364, T366, L368, K370, N390, K392, T394, D399, D401, F405, Y407, K409, T411, and K439, numbered according to the EU numbering system. In certain embodiments, one polypeptidechain of the antibody heavy chain constant region comprises K360E and K409W substitutions relative to SEQ ID NO: 118; and the other polypeptide chain of the antibody heavy chain constant region comprises Q347R, D399V and F405T substitutions relative to SEQ ID NO: 118, numbered according to the EU numbering system. In certain embodiments, one polypeptide chain of the antibody heavy chain constant region comprises an F405L substitution relative to SEQ ID NO: 118; and the other polypeptide chain of the antibody heavy chain constant region comprises a K409R substitution relative to SEQ ID NO: 118, numbered according to the EU numbering system. In certain embodiments, one polypeptide chain of the antibody heavy chain constant region comprises a Y349C substitution relative to SEQ ID NO: 118; and the other polypeptide chain of the antibody heavy chain constant region comprises an S354C substitution relative to SEQ ID NO: 118, numbered according to the EU numbering system.

[0030] In another aspect, the present application provides a protein comprising:(a) a first polypeptide comprising the amino acid sequence of SEQ ID NO:270;(b) a second polypeptide comprising the amino acid sequence of SEQ ID NO: 194; and(c) a third polypeptide comprising the amino acid sequence of SEQ ID NO: 195.

[0031] In another aspect, the present application provides a protein comprising:(a) a first polypeptide comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:270;(b) a second polypeptide comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 194; and(c) a third polypeptide comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 195.

[0032] In some embodiments, the protein provided herein comprises:(a) a first polypeptide comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO:270;(b) a second polypeptide comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 194; and(c) a third polypeptide comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 195.

[0033] In some embodiments, the protein provided herein comprises:(a) a first polypeptide comprising an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:270;(b) a second polypeptide comprising an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 194; and(c) a third polypeptide comprising an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 195.

[0034] In some embodiments, the protein provided herein comprises:(a) a first polypeptide comprising an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO:270;(b) a second polypeptide comprising an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 194; and(c) a third polypeptide comprising an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 195.

[0035] In some embodiments, the protein provided herein comprises:(a) a first polypeptide comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO:270;(b) a second polypeptide comprising the amino acid sequence of SEQ ID NO: 194; and(c) a third polypeptide comprising the amino acid sequence of SEQ ID NO: 195.

[0036] In some embodiments, the protein provided herein comprises:(a) a first polypeptide comprising an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:270;(b) a second polypeptide comprising the amino acid sequence of SEQ ID NO: 194; and(c) a third polypeptide comprising the amino acid sequence of SEQ ID NO: 195.

[0037] In some embodiments, the protein provided herein comprises:(a) a first polypeptide comprising an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO:270;(b) a second polypeptide comprising the amino acid sequence of SEQ ID NO: 194; and(c) a third polypeptide comprising the amino acid sequence of SEQ ID NO: 195.

[0038] In some embodiments, the protein provided herein comprises a polypeptide comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:270. In some embodiments, the protein provided herein comprises a polypeptide comprising an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:270. In some embodiments, the protein provided herein comprises a polypeptide comprising an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO:270.

[0039] In some embodiments, the protein provided herein comprises a polypeptide comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 194. In some embodiments, the protein provided herein comprises a polypeptide comprising an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 194. In some embodiments, the protein provided herein comprises a polypeptide comprising an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 194.

[0040] In some embodiments, the protein provided herein comprises a polypeptide comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 195. In some embodiments, the protein provided herein comprises a polypeptide comprising an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 195. In some embodiments, the protein provided herein comprises a polypeptide comprising an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 195.

[0041] In another aspect, the present application provides a protein comprising: (a) a first polypeptide comprising the amino acid sequence of SEQ ID NO:271;(b) a second polypeptide comprising the amino acid sequence of SEQ ID NO:272; and(c) a third polypeptide comprising the amino acid sequence of SEQ ID NO:273.

[0042] In another aspect, the present application provides a pharmaceutical composition comprising a protein disclosed herein and a pharmaceutically acceptable carrier.

[0043] In another aspect, the present application provides a cell comprising one or more nucleic acids encoding a protein disclosed herein.

[0044] In another aspect, the present application provides a method of enhancing tumor cell death, the method comprising exposing the tumor cell and a natural killer cell to an effective amount of a protein disclosed herein or a pharmaceutical composition disclosed herein.

[0045] In another aspect, the present application provides a method of treating cancer, the method comprising administering to a subject in need thereof an effective amount of a protein disclosed herein or a pharmaceutical composition disclosed herein. In some embodiments, the cancer is selected from the group consisting of B-cell non-Hodgkin’s lymphoma (B-NHL), chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), marginal zone lymphoma, mucosa-associated lymphoid tissue (MALT) lymphoma, primary mediastinal B-cell lymphoma, and acute lymphocytic leukemia (ALL).

[0046] In another aspect, the present application provides a method of enhancing B cell death, the method comprising exposing the B cell and a natural killer cell to an effective amount of a protein disclosed herein or a pharmaceutical composition disclosed herein.

[0047] In another aspect, the present application provides a method of treating an autoimmune inflammatory disease, the method comprising administering to a subject in need thereof an effective amount of a protein disclosed herein or a pharmaceutical composition disclosed herein.

[0048] In some embodiments of a protein disclosed herein, the protein is a purified protein. In some embodiments, the protein is purified using a method selected from the group consisting of: centrifugation, depth filtration, cell lysis, homogenization, freeze-thawing, affinity purification, gel filtration, ion exchange chromatography, hydrophobic interaction exchange chromatography, and mixed-mode chromatography.BRIEF DESCRIPTION OF THE DRAWINGS

[0049] FIG. 1 is a representation of a heterodimeric, multispecific antibody, e.g., a trispecific binding protein (TriNKET). Each arm can represent either the NKG2D binding domain, or the BAFF-R binding domain. In some embodiments, the NKG2D binding domain and the BAFF-R binding domains can share a common light chain.

[0050] FIG. 2A-FIG. 2E illustrate five exemplary formats of a multispecific binding protein, e.g., a trispecific binding protein (TriNKET). As shown in FIG. 2A, either the NKG2D- binding domain or the BAFF-R binding domain can take the scFv format (left arm). An antibody that contains a NKG2D targeting scFv, a BAFF-R targeting Fab fragment, and a heterodimerized antibody constant region is referred herein as the F3-TriNKET. An antibody that contains a BAFF-R targeting scFv, a NKG2D targeting Fab fragment, and a heterodimerized antibody constant region / domain that binds CD 16 is referred herein as the F3’ -TriNKET (FIG. 2E). As shown in FIG. 2B, both the NKG2D binding domain and BAFF-R binding domain can take the scFv format. FIG. 2C-FIG. 2D are illustrations of an antibody with three antigen-binding sites, including two antigen-binding sites that bind BAFF-R, and the NKG2D-binding site fused to the heterodimerized antibody constant region. These antibody formats are referred herein as F4- TriNKET. FIG. 2C illustrates that the two BAFF-R binding sites are in the Fab fragment format, and the NKG2D binding site in the scFv format. FIG. 2D illustrates that the BAFF-R binding sites are in the scFv format, and the NKG2D binding site is in the scFv format. FIG. 2E represents a trispecific antibody (TriNKET) that contains a BAFF-R targeting scFv, a NKG2D targeting Fab fragment, and a heterodimerized antibody constant region / domain (“CD domain”) that binds CD 16. The antibody format is referred herein as F3’ -TriNKET. In certain exemplary multispecific binding proteins, heterodimerization mutations on the antibody constant region include K360E and K409W on one constant domain; and Q347R, D399V and F405T on the opposite constant domain (shown as a triangular lock-and-key shape in the CD domains). The bold bar between the heavy and the light chain variable domains of the Fab fragments represents a disulfide bond.

[0051] FIG. 3 is a representation of a TriNKET in the Triomab form, which is a trifunctional, bispecific antibody that maintains an IgG-like shape. This chimera consists of two half antibodies, each with one light and one heavy chain, that originate from two parentalantibodies. Triomab form may be a heterodimeric construct containing 1 / 2 of rat antibody and 1 / 2 of mouse antibody.

[0052] FIG. 4 is a representation of a TriNKET in the KiH Common Light Chain form, which involves the knobs-into-holes (KIHs) technology. KiH is a heterodimer containing 2 Fab fragments binding to target 1 and 2, and an Fc stabilized by heterodimerization mutations. TriNKET in the KiH format may be a heterodimeric construct with 2 Fab fragments binding to target 1 and target 2, containing two different heavy chains and a common light chain that pairs with both heavy chains.

[0053] FIG. 5 is a representation of a TriNKET in the dual-variable domain immunoglobulin (DVD-Ig™) form, which combines the target-binding domains of two monoclonal antibodies via flexible naturally occurring linkers, and yields a tetravalent IgG-like molecule. DVD-Ig™ is a homodimeric construct where variable domain targeting antigen 2 is fused to the N-terminus of a variable domain of Fab fragment targeting antigen 1. DVD-Ig™ form contains normal Fc.

[0054] FIG. 6 is a representation of a TriNKET in the Orthogonal Fab fragment interface (Ortho-Fab) form, which is a heterodimeric construct that contains 2 Fab fragments binding to target 1 and target 2 fused to Fc. Light chain (LC)-heavy chain (HC) pairing is ensured by orthogonal interface. Heterodimerization is ensured by mutations in the Fc.

[0055] FIG. 7 is a representation of a TriNKET in the 2-in-l Ig format.

[0056] FIG. 8 is a representation of a TriNKET in the ES form, which is a heterodimeric construct containing two different Fab fragments binding to target 1 and target 2 fused to the Fc. Heterodimerization is ensured by electrostatic steering mutations in the Fc.

[0057] FIG. 9 is a representation of a TriNKET in the Fab Arm Exchange form: antibodies that exchange Fab fragment arms by swapping a heavy chain and attached light chain (halfmolecule) with a heavy-light chain pair from another molecule, resulting in bispecific antibodies. Fab Arm Exchange form (cFae) is a heterodimer containing 2 Fab fragments binding to target 1 and 2, and an Fc stabilized by heterodimerization mutations.

[0058] FIG. 10 is a representation of a TriNKET in the SEED Body form, which is a heterodimer containing 2 Fab fragments binding to target 1 and 2, and an Fc stabilized by heterodimerization mutations.

[0059] FIG. 11 is a representation of a TriNKET in the LuZ-Y form, in which a leucine zipper is used to induce heterodimerization of two different HCs. The LuZ-Y form is a heterodimer containing two different scFabs binding to target 1 and 2, fused to Fc. Heterodimerization is ensured through leucine zipper motifs fused to C-terminus of Fc.

[0060] FIG. 12 is a representation of a TriNKET in the Cov-X-Body form.

[0061] FIG. 13A and FIG. 13B are representations of TriNKETs in the ick-Body forms, which are heterodimeric constructs with two different Fab fragments fused to Fc stabilized by heterodimerization mutations: one Fab fragment targeting antigen 1 contains kappa LC, and the second Fab fragment targeting antigen 2 contains lambda LC. FIG. 13A is an exemplary representation of one form of a KX-Body; FIG. 13B is an exemplary representation of another KX- Body.

[0062] FIG. 14 is a representation of an OAsc-Fab heterodimeric construct that includes Fab fragment binding to target 1 and scFab binding to target 2, both of which are fused to the Fc domain. Heterodimerization is ensured by mutations in the Fc domain.

[0063] FIG. 15 is a representation of a DuetMab, which is a heterodimeric construct containing two different Fab fragments binding to antigens 1 and 2, and an Fc that is stabilized by heterodimerization mutations. Fab fragments 1 and 2 contain differential S-S bridges that ensure correct light chain and heavy chain pairing.

[0064] FIG. 16 is a representation of a CrossmAb, which is a heterodimeric construct with two different Fab fragments binding to targets 1 and 2, and an Fc stabilized by heterodimerization mutations. CL and CHI domains, and VH and VL domains are switched, e.g., CHI is fused in-line with VL, and CL is fused in-line with VH.

[0065] FIG. 17 is a representation of a Fit-Ig, which is a homodimeric construct where Fab fragment binding to antigen 2 is fused to the N-terminus of HC of Fab fragment that binds to antigen 1. The construct contains wild-type Fc.

[0066] FIG. 18A-FIG. 18C are line graphs showing binding of BAFF-R-targeting TriNKETs derived from hCOH-2 (FIG. 18A), Genentech Hu9.1-73 (FIG. 18B), and ianalumab- based antigen-binding site (the three versions, F3’, 2-Fab, and ianalumab-mAb, do not contain antibody-dependent cellular cytotoxicity-enhancing mutations present in the commercial ianalumab antibody) (FIG. 18C) to BAFF-R-positive RAJI cells.

[0067] FIG. 19A-FIG. 19C are line graphs showing NK cell-mediated lysis of BAFF-R- positive RAJI cells by primary NK cells in the presence of BAFF-R-targeting TriNKETs derived from hCOH-2 (FIG. 19A), Genentech Hu9.1-73 (FIG. 19B), and ianalumab-based antigenbinding site (the three versions, F3’, 2-Fab, and ianalumab-mAb, do not contain antibodydependent cellular cytotoxicity-enhancing mutations present in the commercial ianalumab antibody) (FIG. 19C).

[0068] FIG. 20A-FIG. 20C are line graphs showing NK cell-mediated lysis of BAFF-R- positive RAJI cells by KHYG-CD16V cells in the presence of BAFF-R-targeting TriNKETs derived from hCOH-2 (FIG. 20A), Genentech Hu9.1-73 (FIG. 20B), and ianalumab-based antigen-binding site (the three versions, F3’, 2-Fab, and ianalumab-mAb, do not contain antibody-dependent cellular cytotoxicity-enhancing mutations present in the commercial ianalumab antibody) (FIG. 20C).

[0069] FIG. 21 is a graph showing fluorescence outputs from a blocking assay of BAFF- biotin binding to hBAFF-R expressed on CHO cells by the indicated antibodies.

[0070] FIG. 22A-FIG. 22D are graphs of fluorescence outputs from binding assays on CHO cells showing binding of indicated antibodies to hBAFF-R (FIG. 22A, FIG. 22B) or blocking assays of BAFF-biotin binding to BAFF-R by indicated antibodies (FIG. 22C, FIG. 22D).

[0071] FIG. 23A-FIG. 23E are flow cytometry plots showing binding of AB0369scFv expressed on yeast to no antigen control (FIG. 23 A), h-BAFF-R-hFc (FIG. 23B), Irrel evant-hFc (FIG. 23 C), hBAFF-R-GST (FIG. 23D), or Irrelevant-GST (FIG. 23E). Vertical axes indicates scFv expression as measured by detection of the Flag epitope tag; horizonal axes indicate binding of biotinylated control of BAFF-R constructs to scFv as measured by detection of streptavidin-PE.

[0072] FIG. 24A and FIG. 24B are graphs showing binding of AB0369 or indicated controls to human (FIG. 24A) or cynomolgus monkey (FIG. 24B) BAFF-R.

[0073] FIG. 25A-FIG. 25G detail a poly-specificity assay of a multi-specific binding proteins with a BAFF-R binding site derived from AB0369. FIG. 25A is a schematic of the assay. FIG. 25B-FIG. 25G show graphs of AB0369 (left panels), trastuzumab negative control (middle panels), or ixekizumab positive control (right panels) in the absence (top panels) or presence (bottom panels) of poly-specificity reagent (PSR).

[0074] FIG. 26 is a graph showing a KHYG-l-CD16aV cytotoxicity assay of Ramos cells as induced by a multispecific binding protein with a BAFF-R binding site derived from AB0369.

[0075] FIG. 27 is a graph showing fluorescence outputs from a binding assay showing blockage of BAFF-biotin binding to human BAFF-R expressed on CHO cells by AB0369 or indicated.

[0076] FIG. 28A-FIG. 28D are flow cytometry plots showing binding of hBAFF-R-hFc- His to parental AB0369scFv or clones selected from a library produced by affinity maturation expressed on yeast following successive rounds of selection. FIG. 28A shows binding to parental AB0369scFv; FIG. 28B shows binding to sample from the first round of clone selection; FIG. 28C shows binding to sample from the second round of clone selection; FIG. 28D shows binding to output from the second round of clone selection.

[0077] FIG. 29A-FIG. 29E are flow cytometry plots showing binding of hBAFF-R-hFc- His to AB0369 and affinity-matured scFv clones expressed on yeast. FIG. 29A shows binding to parental AB0369; FIG. 29B shows binding to AB0605; FIG. 29C shows binding to AB0622; FIG. 29D shows binding to AB0622; and FIG. 29E shows binding to ianalumab-based antigenbinding site.

[0078] FIG. 30A-FIG. 30C are graphs demonstrating BAFF-R binding and cytotoxicity of multi-specific binding proteins developed from affinity maturation of AB0369. FIG. 30A is a graph showing binding of multi-specific binding proteins with BAFF-R binding sites derived from indicated clones to human BAFF-R expressed on CHO cells. FIG. 30B is a graph showing a KHYG-l-CD16aV cytotoxicity assay of Ramos cells as induced by multi-specific binding proteins with BAFF-R binding sites derived from indicated clones. FIG. 30C is a graph showinga KHYG-l-CD16aV cytotoxicity assay of Ramos cells as induced by multi-specific binding proteins with BAFF-R binding sites derived from AB0622.

[0079] FIG. 31A-FIG. 31E detail a poly-specificity assay of multi-specific binding proteins with BAFF-R binding sites derived from AB00605 and AB0606. FIG. 31 A is a schematic of the assay. FIG. 31B-FIG. 3 IE show graphs of AB0605 (left panels) or AB0606 (right panels) in the absence (top panels) or presence (bottom panels) of poly-specificity reagent (PSR).

[0080] FIG. 32A-FIG. 32C are flow cytometry plots showing binding of hBAFF-R-hFc- His to parental AB0369scFv or clones selected from a library produced by affinity maturation and expressed on yeast following successive rounds of selection. FIG. 32A shows binding to parental AB0369scFv; FIG. 32B shows binding to sample from the first round of clone selection; FIG. 32C shows binding to sample from the second round of clone selection.

[0081] FIG. 33A-FIG. 33E are flow cytometry plots showing binding of hBAFF-R-hFc- His to AB0369 and affinity-matured scFv clones expressed on yeast. FIG. 33A shows binding to parental AB0369; FIG. 33B shows binding to AB0679; FIG. 33C shows binding to AB0681; FIG. 33D shows binding to AB0682; and FIG. 33E shows binding to ianalumab-based antigenbinding site.

[0082] FIG. 34A-FIG. 34C are graphs demonstrating BAFF-R binding to multi-specific binding proteins developed from affinity maturation of AB0369. FIG. 34A is a graph showing binding of multi-specific binding proteins with BAFF-R binding sites derived from indicated clones to human BAFF-R expressed on CHO cells. FIG. 34B is a graph showing binding of multi-specific binding proteins with BAFF-R binding sites derived from indicated clones to cynomolgus monkey BAFF-R expressed on CHO cells. FIG. 34C is a graph showing fluorescence outputs from a binding assay showing blockage of BAFF-biotin binding to BAFF-R expressed on CHO cells by the indicated antibodies.

[0083] FIG. 35 is a graph showing a KHYG-l-CD16aV cytotoxicity assay of BJAB cells as induced by multi-specific binding proteins with BAFF-R binding sites derived from AB0679, AB0568, or Tool-F3’ positive control.

[0084] FIG. 36A-FIG. 36D are flow cytometry plots showing binding of hBAFF-R-hFc- His to parental AB0369scFv clones selected from a library produced by affinity maturation expressed on yeast following successive rounds of selection. FIG. 36A shows binding to parental AB0369scFv; FIG. 36B shows binding to sample from the first round of clone selection; FIG. 36C shows binding to sample from the second round of clone selection; and FIG. 36D shows binding to sample from the third round of clone selection.

[0085] FIG. 37A-FIG. 37F are flow cytometry plots showing binding of hBAFF-R-hFc- His to AB0369 and affinity-matured scFv clones expressed on yeast. FIG. 37A shows binding to parental AB0369; FIG. 37B shows binding to AB0682; FIG. 37C shows binding to AB0898; FIG. 37D shows binding to AB0899; FIG. 37E shows binding to AB0900; and FIG. 37F shows binding to ianalumab-based antigen-binding site.

[0086] FIG. 38 is a graph showing a KHYG-l-CD16aV cytotoxicity assay of BJAB cells as induced by multi-specific binding proteins with BAFF-R binding sites derived from AB0898, AB0899, or AB0900.

[0087] FIG. 39A-FIG. 39C show graphs of differential scanning calorimetry (DSC) profiles of AB0898 (FIG. 39A), AB0899 (FIG. 39B), and AB0900 (FIG. 39C).

[0088] FIG. 40 shows flow cytometry plots of binding of scFv clones expressed on yeast to biotinylated hBAFFR-Fc before (left) and after (right) challenge by incubation with 1 mM nonbiotinylated hBAFFR-Fc.

[0089] FIG. 41A and FIG. 41B show flow cytometry plots of binding of scFv clones expressed on yeast to biotinylated hBAFFR-Fc before (FIG. 41 A) and after (FIG. 41B) challenge by incubation with 1 mM non-biotinylated hBAFFR-Fc. Clones tested are (left-to-right) AB1080, AB1081, AB1084, AB1085, and ianalumab.

[0090] FIG. 42A and FIG. 42B are graphs showing binding of indicated antibody clones to human (FIG. 42 A) or cynomolgus monkey (FIG. 42B) BAFF-R.

[0091] FIG. 43A-FIG. 431 detail a poly-specificity assay of a multi-specific binding proteins with a BAFF-R binding site derived from AB1080 or AB1081. FIG. 43A is a schematic of the assay. FIG. 43B-FIG. 431 show graphs of AB 1080 (left panels), AB 1081 (middle-leftpanels), trastuzumab negative control (middle-right panels), or ixekizumab positive control (right panels) in the absence (top panels) or presence (bottom panels) of poly-specificity reagent (PSR).

[0092] FIG. 44A and FIG. 44B show graphs of a KHYG-l-CD16aV cytotoxicity assay of BJAB cells as induced by multi-specific binding proteins with BAFF-R binding sites derived from AB1080 (FIG. 44A) or AB1085 (FIG. 44B) compared to Tool positive control.

[0093] FIG. 45 is a graph showing fluorescence outputs from a blocking assay of BAFF- biotin binding to human BAFF-R expressed on CHO cells by the indicated antibody clones.

[0094] FIG. 46A-FIG. 46D show graphs of nano-dual scanning fluorimetry (nanoDSF) analysis of multi-specific binding proteins with BAFF-R binding sites derived from AB1080 (FIG. 46A), AB 1081 (FIG. 46B), AB 1084 (FIG. 46C), and AB 1085 (FIG. 46D).

[0095] FIG. 47 shows a graph of hydrophobic interaction chromatography (HIC) analysis of multi-specific binding proteins with BAFF-R binding sites derived from indicated antibodies.

[0096] FIG. 48 shows a graph of HIC analysis of AB 1612 compared to indicated benchmark biologies.

[0097] FIG. 49A and FIG. 49B are graphs showing binding of indicated antibody clones to cynomolgus monkey (FIG. 49 A) or human (FIG. 49B) BAFF-R.

[0098] FIG. 50 is a graph showing fluorescence outputs from a binding assay showing blockage of BAFF-biotin binding to human BAFF-R expressed on CHO cells by the indicated antibodies.

[0099] FIG. 51A-FIG. 51C show the surface charge distribution of the BAFF-R binding arm of AB1424 / 1612 F3’ TriNKET. Three orientations are shown: both facades (left panel: front view; center panel: back view) and the antigen-engaging surface (right panel: top view). The positively charged areas are colored blue, negatively charged areas red, and the hydrophobic surface white.

[0100] FIG. 52A-FIG. 52E are graphs showing evaluation of surface patches and CDRs length of the BAFF-R binding arm of AB1424 / 1612 F3’ TriNKET. Solid lines and corresponding arrows indicate the scoring of the BAFF-R binding arm of AB1424 / 1612 F3’ TriNKET in reference to a database of 377 late-stage therapeutic antibodies. In FIG. 52A and FIG. 52B, the two inner dashed lines indicate 2 standard deviations (>95% of referencemolecules within this region), whereas the two outer most dashed lines indicate 3 standard deviations (>99.7% of reference molecules within this region). In each plot of FIG. 52C-FIG. 52E, there are two dashed lines - one closer and the other further to the solid line. The dashed line closer to the solid line indicates 2 standard deviations (>95% of reference molecules within this region), whereas the dashed line further to the solid line indicates 3 standard deviations (>99.7% of reference molecules within this region).

[0101] FIG. 53A-FIG. 53C show the surface charge distribution of the NKG2D binding arm of AB1424 / 1612 F3’ TriNKET. Three orientations are shown: both facades (left panel: front view; center panel: back view) and the antigen-engaging surface (right panel: top view). The positively charged areas are colored blue, negatively charged areas red, and the hydrophobic surface white.

[0102] FIG. 54A-FIG. 54E are graphs showing evaluation of surface patches and CDRs length of the NKG2D-R binding arm of AB 1424 / 1612 F3’ TriNKET. Solid lines and corresponding arrows indicate the scoring of the BAFF-R binding arm of AB1424 / 1612 F3’ TriNKET in reference to a database of 377 late-stage therapeutic antibodies. In FIG. 54A and FIG. 54B, the two inner dashed lines indicate 2 standard deviations (>95% of reference molecules within this region), whereas the two outer most dashed lines indicate 3 standard deviations (>99.7% of reference molecules within this region). In each plot of FIG. 54C-FIG. 54E, there are two dashed lines - one closer and the other further to the solid line. The dashed line closer to the solid line indicates 2 standard deviations (>95% of reference molecules within this region), whereas the dashed line further to the solid line indicates 3 standard deviations (>99.7% of reference molecules within this region).

[0103] FIG. 55A and FIG. 55B are chromatograms showing HIC analysis of AB1424 / 1612 F3’ TriNKET (FIG. 55A) and comparison with adalimumab and pembrolizumab (FIG. 55B).

[0104] FIG. 56 is a graph showing capillary isoelectric focusing (cIEF) profiling of AB1424 / 1612 F3’ TriNKET.

[0105] FIG. 57A and FIG. 57B are graphs showing DSC profiling of AB1424 / 1612 F3’ TriNKET in PBS pH 7.4 (FIG. 57A) and HST pH 6.0 (FIG. 57B).

[0106] FIG. 58A and FIG. 58B are graphs showing n-curve analysis (FIG. 58A) and confidence interval (FIG. 58B) of AB 1424 / 1612 F3’ TriNKET binding cell-based BAFF-R by Kinexa.

[0107] FIG. 59A and FIG. 59B are graphs showing binding of AB1424 / 1612 F3’ TriNKET and corresponding parental mAb to isogenic human (FIG. 59A) and cynomolgus (FIG. 59B) BAFF-R-CHO cells.

[0108] FIG. 60A-FIG. 60F are graphs showing binding of AB1424 / 1612 F3’ TriNKET to BAFF-R+ tumor cell lines. Titrations were done in the presence of BJAB (FIG. 60A), Raji (FIG. 60B), RL (FIG. 60C), Rs4;l l (FIG. 60D), Jeko-1 (FIG. 60E), SUDHL-6 cells (FIG. 60F). FOB = fold over background of stained vs. unstained samples.

[0109] FIG. 61A-FIG. 61H are graphs showing surface plasmon resonance (SPR) binding of AB1424 / 1612 F3’ TriNKET to human NKG2D. Colored lines represent raw data and black traces represent 1 : 1 binding fit (top panel). Corresponding steady state fits (bottom panel). The vertical line denotes steady state KD.

[0110] FIG. 62A-FIG. 62H are graphs showing SPR binding of AB1424 / 1612 F3’ TriNKET to cynomolgus NKG2D. Colored lines represent raw data and black traces represent 1 : 1 binding fit (top panel). Corresponding steady state fits (bottom panel). The vertical line denotes steady state KD.

[0111] FIG. 63A-FIG. 63H are graphs showing SPR binding of AB1424 / 1612 F3’ TriNKET to human CD16a V158 (top panels) or trastuzumab (bottom panels). Colored lines represent raw data and black traces represent 1 : 1 binding fit.

[0112] FIG. 64A-FIG. 64P are graphs showing SPR binding of AB1424 / 1612 F3’ TriNKET (top panels) or trastuzumab (bottom panels) to human CD16a F158. Colored lines represent raw data and black traces represent 1 : 1 binding fit (top panel).

[0113] FIG. 65A-FIG. 65H are graphs showing SPR binding of AB1424 / 1612 F3’ TriNKET to cynomolgus CD 16. Colored lines represent raw data and black traces represent 1 : 1 binding fit (top panel). Corresponding steady state fits (bottom panel). The vertical line denotes steady state KD.

[0114] FIG. 66 is a graph showing SPR binding of AB1424 / 1612 F3’ TriNKET to NKG2D (brown), CD 16a (purple), or mixed CD 16a and NKG2D (blue) surfaces.

[0115] FIG. 67A and FIG. 67B are sensorgram graphs representing binding of BAFF-R (800 nM) followed by binding of hNKG2D (7 pM) to captured AB1424 / 1612 F3’ TriNKET (FIG. 67 A) or reverse order of target binding with human NKG2D (7pM) followed by BAFF-R (800 nM) (FIG. 67B).

[0116] FIG. 68A and FIG. 68B are graphs showing SPR analysis of BAFF-R and TACI binding to immobilized AB1424 / 1612 F3’ TriNKET (FIG. 68A) and specific anti-TACI mAb (FIG. 68B).

[0117] FIG. 69A and FIG. 69B are graphs showing binding of AB1424 / 1612 F3’ TriNKET to parental cells not expressing BCMA (FIG. 69A) and isogenic BCMA+ cells compared to control mAb specific anti-BCMA (FIG. 69B).

[0118] FIG. 70A and FIG. 70B are graphs showing AB 1424 / 1612 F3’ TriNKET binding to isogenic BAFFR+ CHO cells (FIG. 70A) and lack of reactivity with parental CHO line (FIG. 70B).

[0119] FIG. 71A-FIG. 71G detail a poly-specificity assay of a AB1424 / 1612 F3’ TriNKET. FIG. 71A is a schematic of the assay. FIG. 71B-FIG. 71G show graphs of AB1424 / 1612 F3’ TriNKET (left panels), trastuzumab negative control (middle panels), or ixekizumab positive control (right panels) in the absence (top panels) or presence (bottom panels) of poly-specificity reagent (PSR).

[0120] FIG. 72A-FIG. 72C show graphs of cytotoxicity assays of RL cells as induced by AB1424 / 1612 F3’ TriNKET (blue) or parental monoclonal antibody (red) using NK cells from three donors.

[0121] FIG. 73A-FIG. 73D show schematic representations of AB 1424 / 1612 F3’TriNKET and controls for elucidating mechanism of action.

[0122] FIG. 74 shows a graph of a KHYG-l-CD16aV cytotoxicity assay of BJAB cells as induced by AB1424 / 1612 F3’ TriNKET (blue), AB1424 / 1612 F3’ TriNKET lacking NKG2D binding (black), or AB1424 / 1612 F3’ TriNKET- Fc silenced (red), or palivizumab F3’ TriNKET (grey).

[0123] FIG. 75A-FIG. 75H are sensorgram graphs showing binding of AB 1424 / 1612 F3’ TriNKET (top panels) and trastuzumab (bottom panels) to human CD64. Raw sensorgrams (colored) with 1 : 1 fitted curves overlaid (black).

[0124] FIG. 76A-FIG. 76H are sensorgram graphs showing binding of AB 1424 / 1612 F3’ TriNKET (top panels) and trastuzumab (bottom panels) to cynomolgus monkey CD64. Raw sensorgrams (colored) with 1 : 1 fitted curves overlaid (black).

[0125] FIG. 77A-FIG. 77P are sensorgram graphs showing binding of AB1424 / 1612 F3’ TriNKET (FIG. 77A-FIG. 77H) and trastuzumab (FIG. 77I-FIG. 77P) to human CD32a H131. For each molecule the upper panel represents raw sensorgrams and the lower panel represents the steady state affinity fit.

[0126] FIG. 78A-FIG. 78P are sensorgram graphs showing binding of AB 1424 / 1612 F3’ TriNKET (FIG. 78A-FIG. 78H) and trastuzumab (FIG. 78I-FIG. 78P) to human CD32a R131. For each molecule the upper panel represents raw sensorgrams and the lower panel represents the steady state affinity fit.

[0127] FIG. 79A-FIG. 79P are sensorgram graphs showing binding of AB 1424 / 1612 F3’ TriNKET (FIG. 79A-FIG. 79H) and trastuzumab (FIG. 79I-FIG. 79P) to human CD32b. For each molecule the upper panel represents raw sensorgrams and the lower panel represents the steady state affinity fit.

[0128] FIG. 80A-FIG. 80P are sensorgram graphs showing binding of AB1424 / 1612 F3’ TriNKET (FIG. 80A-FIG. 80H) and trastuzumab (FIG. 80I-FIG. 80P) to human CD 16b. For each molecule the upper panel represents raw sensorgrams and the lower panel represents the steady state affinity fit.

[0129] FIG. 81A-FIG. 81H are sensorgram graphs showing binding of AB1424 / 1612 F3’ TriNKET (top panels) and trastuzumab (bottom panels) to cynomolgus monkey CD 16.

[0130] FIG. 82A-FIG. 82P are sensorgram graphs showing binding of AB 1424 / 1612 F3’ TriNKET (FIG. 82A-FIG. 82H) and trastuzumab (FIG. 82I-FIG. 82P) to human FcRn at pH 6.0. For each molecule the upper panel represents raw sensorgrams and the lower panel represents the steady state affinity fit.

[0131] FIG. 83A-FIG. 83P are sensorgram graphs showing binding of AB 1424 / 1612 F3’ TriNKET (FIG. 83 A-FIG. 83H) and trastuzumab (FIG. 83I-FIG. 83P) to cynomolgus monkey FcRn at pH 6.0. For each molecule the upper panel represents raw sensorgrams and the lower panel represents the steady state affinity fit.

[0132] FIG. 84A-FIG. 84H are raw sensorgram graphs showing binding of AB 1424 / 1612 F3’ TriNKET (top panels) and trastuzumab (bottom panels) to human (left panels) and cynomolgus monkey (right panels) FcRn at pH 7.4.

[0133] FIG. 85 shows a graph of a KHYG-l-CD16aV cytotoxicity assay of BJAB cells as induced by two lots of AB1424 / 1612 F3’ TriNKET (blue and red) or human IgGlk (grey).

[0134] FIG. 86A shows a graph of a KHYG-l-CD16aV cytotoxicity assay of BJAB cells as induced by two lots of AB1424 / 1612 F3’ TriNKET (blue and red) or human IgGlk (grey).

[0135] FIG. 86B shows a graph of a KHYG-l-CD16aV cytotoxicity assay of BJAB cells as induced by AB1424 / 1612 F3’ TriNKET at nominal drug concentrations (NDC) of 50% (red), 100% (blue), and 200% (green).

[0136] FIG. 87A and FIG. 87B show PEG precipitation Cm plots of AB1424 / 1612 F3’ TriNKET in histidine (FIG. 87A) and acetate (FIG. 87B).

[0137] FIG. 88A and FIG. 88B show PEG precipitation Cm plots of adalimumab in histidine (FIG. 88A) and acetate (FIG. 88B).

[0138] FIG. 89A-FIG. 89C show ko plots of adalimumab in acetate (FIG. 89A), histidine (FIG. 89B), and phosphate (FIG. 89C).

[0139] FIG. 90A-FIG. 90C show kDplots of AB1424 / 1612 F3’ TriNKET in acetate (FIG. 90A), histidine (FIG. 90B), and phosphate (FIG. 90C).

[0140] FIG. 91 is a viscosity vs. concentration plot of AB1424 / 1612 F3’ TriNKET at 25°C.

[0141] FIG. 92 is a chromatogram of size-exclusion chromatography (SEC) analysis of AB1424 / 1612 F3’ TriNKET after 4 weeks at 40 °C in HST, pH 6.0 compared to control.

[0142] FIG. 93 is a graph of capillary electrophoresis sodium dodecyl sulfate (CE-SDS) analysis of AB1424 / 1612 F3’ TriNKET after 4 weeks at 40 °C in HST, pH 6.0 compared to control.

[0143] FIG. 94 is a graph showing cIEF profiling of AB1424 / 1612 F3’ TriNKET in HST, pH 6.0 compared to control.

[0144] FIG. 95A-FIG. 95C show binding of AB1424 / 1 612 F3’ TriNKET to hBAFF-R, hNKG2D and hCD16aV after 4 weeks at 40°C in HST, pH 6.0 compared to control. FIG. 95A is a graph showing binding to BJAB cells (BAFF-R); FIG. 95B is a sensorgram showing binding to hNKG2D by SPR. FIG. 95C is a sensorgram showing binding to hCD16a V158 by SPR.Colored sensorgrams represent raw data and black overlays represent the kinetic fit of the raw data.

[0145] FIG. 96 shows a graph of a KHYG-l-CD16aV cytotoxicity assay of BJAB cells as induced by AB1424 / 1612 F3’ TriNKET after 1 week (red), 2 weeks (green), 3 weeks (purple) at 40°C in HST, pH 6.0 compared to control (blue).

[0146] FIG. 97A-FIG. 97C show the surface charge distribution of the BAFF-R binding arm of AB1424 / 1612 F4 TriNKET. Three orientations are shown: both facades (left panel: front view; center panel: back view) and the antigen-engaging surface (right panel: top view). The positively charged areas are colored blue, negatively charged areas red, and the hydrophobic surface white.

[0147] FIG. 98A-FIG. 98E are graphs showing evaluation of surface patches and CDRs length of the BAFF-R binding arm of AB1424 / 1612 F4 TriNKET. Solid lines and corresponding arrows indicate the scoring of the BAFF-R binding arm of AB 1424 / 1612 F4 TriNKET in reference to a database of 377 late-stage therapeutic antibodies. In FIG. 98A and FIG. 98B, the two inner dashed lines indicate 2 standard deviations (>95% of reference molecules within this region), whereas the two outer most dashed lines indicate 3 standard deviations (>99.7% of reference molecules within this region). In each plot of FIG. 98C-FIG. 98E, there are two dashed lines - one closer and the other further to the solid line. The dashed line closer to the solid line indicates 2 standard deviations (>95% of reference molecules within this region), whereas the dashed line further to the solid line indicates 3 standard deviations (>99.7% of reference molecules within this region).

[0148] FIG. 99A-FIG. 99C shows the surface charge distribution of the NKG2D binding arm of AB1424 / 1612 F4 TriNKET. Three orientations are shown: both facades (left panel: front view; center panel: back view) and the antigen-engaging surface (right panel: top view). Thepositively charged areas are colored blue, negatively charged areas red, and the hydrophobic surface white.

[0149] FIG. 100A-FIG. 100E are graphs showing evaluation of surface patches and CDRs length of the NKG2D-R binding arm of AB1424 / 1612 F4 TriNKET. Solid lines and corresponding arrows indicate the scoring of the BAFF-R binding arm of AB1424 / 1612 F3’ TriNKET in reference to a database of 377 late-stage therapeutic antibodies. In FIG. 100A and FIG. 100B, the two inner dashed lines indicate 2 standard deviations (>95% of reference molecules within this region), whereas the two outer most dashed lines indicate 3 standard deviations (>99.7% of reference molecules within this region). In each plot of FIG. 100C-FIG. 100E, there are two dashed lines - one closer and the other further to the solid line. The dashed line closer to the solid line indicates 2 standard deviations (>95% of reference molecules within this region), whereas the dashed line further to the solid line indicates 3 standard deviations (>99.7% of reference molecules within this region).

[0150] FIG. 101A-FIG. 101C are chromatograms of SEC analysis of three lots of AB1424 / 1612 F4 TriNKET.

[0151] FIG. 102 is a graph showing cIEF profiling of three lots of AB1424 / 1612 F4 TriNKET.

[0152] FIG. 103A and FIG. 103B. FIG. 103A is a graph of HIC analysis of AB1424 / 1612 F4 TriNKET compared in indicated benchmark commercial antibodies. FIG. 103B is a graph of thermal stability analysis of AB1424 / 1612 F4 TriNKET by DSC.

[0153] FIG. 104A and FIG. 104B show extracted ion chromatogram (XICs) for the engineered disulfide pair in the Fc (non-reduced and reduced) and the most intense charge state for that peptide pair.

[0154] FIG. 105A and FIG. 105B show XICs for the engineered disulfide pair in the scFv (non-reduced and reduced) and the most intense charge state for that peptide pair.

[0155] FIG. 106 A and FIG. 106B are graphs showing binding of AB 1424 / 1612 F4 TriNKET, parental mAb, and F4-palivizumab to human (FIG. 106A) and cynomolgus (FIG. 106B) BAFF-R+ isogenic CHO cells.

[0156] FIG. 107A-FIG. 107L are sensorgram graphs of SPR binding of AB1424 / 1612 F4 TriNKET to human NKG2D.

[0157] FIG. 108A-FIG. 108P are sensorgram graphs showing binding of AB 1424 / 1612 F4 TriNKET (FIG. 108A-FIG. 108H) and trastuzumab (FIG 108I-FIG. 108P) to human CD32a R131. For each molecule the upper panel represents raw sensorgrams and the lower panel represents the steady state affinity fit.

[0158] FIG. 109A-FIG. 109H are sensorgram graphs showing binding of AB1424 / 1612 F4 TriNKET (top panels) and trastuzumab (bottom panels) to human CD16a V158. For each molecule the upper panel represents raw sensorgrams and the lower panel represents the steady state affinity fit.

[0159] FIG. 110A-FIG. 110H are sensorgram graphs showing binding of AB1424 / 1612 F4 TriNKET (top panels) and trastuzumab (bottom panels) to human CD16a V158. For each molecule the upper panel represents raw sensorgrams and the lower panel represents the steady state affinity fit.

[0160] FIG. 111A-FIG. 111H are sensorgram graphs showing binding of AB1424 / 1612 F4 TriNKET (top panels) and trastuzumab (bottom panels) to human CD64. Raw sensorgrams (colored) with 1 : 1 fitted curves overlaid (black).

[0161] FIG. 112A-FIG. 112H are sensorgram graphs showing binding of AB1424 / 1612 F4 TriNKET (top panels) and trastuzumab (bottom panels) to cynomolgus CD64. Raw sensorgrams (colored) with 1 : 1 fitted curves overlaid (black).

[0162] FIG. 113A-FIG. 113P are sensorgram graphs showing binding of AB1424 / 1612 F4 TriNKET (FIG. 113A-FIG. 113H) and trastuzumab (FIG. 113I-FIG. 113P) to human CD32a H131. For each molecule the upper panel represents raw sensorgrams and the lower panel represents the steady state affinity fit.

[0163] FIG. 114A-FIG. 114P are sensorgram graphs showing binding of AB 1424 / 1612 F4 TriNKET (FIG. 114A-FIG. 114H) and trastuzumab (FIG. 114I-FIG. 114P) to human CD32b.For each molecule the upper panel represents raw sensorgrams and the lower panel represents the steady state affinity fit.

[0164] FIG. 115A-FIG. 115P are sensorgram graphs showing binding of AB 1424 / 1612 F4 TriNKET (FIG. 115A-FIG. 115H) and trastuzumab (FIG. 115I-FIG. 115P) to human CD 16b. For each molecule the upper panel represents raw sensorgrams and the lower panel represents the steady state affinity fit.

[0165] FIG. 116A-FIG. 116P are sensorgram graphs showing binding of AB 1424 / 1612 F4 TriNKET (FIG. 116A-FIG. 116H) and trastuzumab (FIG. 116I-FIG. 116P) to human FcRn at pH 6.0. For each molecule the upper panel represents raw sensorgrams and the lower panel represents the steady state affinity fit.

[0166] FIG. 117A-FIG. 117P are sensorgram graphs showing binding of AB 1424 / 1612 F4 TriNKET (FIG. 117A-FIG. 117H) and trastuzumab (FIG. 117I-FIG. 117P) to cynomolgus FcRn at pH 6.0. For each molecule the upper panel represents raw sensorgrams and the lower panel represents the steady state affinity fit.

[0167] FIG. 118A-FIG. 118H are raw sensorgram graphs showing binding of AB1424 / 1612 F4 TriNKET (top panels) and trastuzumab (bottom panels) to human (left panels) and cynomolgus (right panels) FcRn at pH 7.4.

[0168] FIG. 119 is a graph showing SPR binding of AB1424 / 1612 F4 TriNKET to NKG2D (brown), CD 16a (purple), or mixed CD 16a and NKG2D (blue) surfaces.

[0169] FIG. 120A and FIG. 120B are graphs showing sequential saturation of BAFF-R and NKG2D by AB1424 / 1612 F4 TriNKET.

[0170] FIG. 121A-FIG. 1211 detail a poly-specificity assay of AB1424 / 1612 F4 TriNKET. FIG. 121 A is a schematic of the assay. FIG. 121B-FIG. 1211 show graphs of AB 1424 / 1612 F4 TriNKET (left panels), trastuzumab (center-left panels), rituximab (center-right panels), or ixekizumab (right panels) in the absence (top panels) or presence (bottom panels) of polyspecificity reagent (PSR).

[0171] FIG. 122 is a graph of a KHYG-l-CD16aV cytotoxicity assay of BJAB cells as induced by AB 1424 / 1612 F4 TriNKET (blue) and human IgGlk (grey).

[0172] FIG. 123 is a graph of a rested hNK-induced cytotoxicity assay of BJAB cells as induced by AB 1424 / 1612 F4 TriNKET (blue) and parental mAb (red).

[0173] FIG. 124 is a chromatogram of SEC analysis of AB1424 / 1612 F4 TriNKET after 4 weeks at 40 °C in HST, pH 6.0 compared to control.

[0174] FIG. 125 is a graph showing reduced CE-SDS analysis of AB1424 / 1612 F4 TriNKET after 4 weeks at 40 °C in HST, pH 6.0 compared to control.

[0175] FIG. 126 is a graph showing cIEF profiling of AB1424 / 1612 F4 TriNKET after 4 weeks at 40 °C in HST, pH 6.0 compared to control.

[0176] FIG. 127 is a graph showing binding of AB1424 / 1612 F4 TriNKET to to hBAFF- R+ cells after 4 weeks at 40 °C in HST, pH 6.0 compared to control.

[0177] FIG. 128A and FIG. 128B are sensorgram graphs showing SPR binding of hCD16aV to AB1424 / 1612 F4 TriNKET after 4 weeks at 40 °C in HST, pH 6.0 (FIG. 128B) compared to control (FIG. 128A).

[0178] FIG. 129 is a graph of a KHYG-l-CD16aV cytotoxicity assay of BJAB cells as induced by AB 1424 / 1612 F4 TriNKET after 4 weeks at 40 °C in HST, pH 6.0 (red) compared to control (blue).

[0179] FIG. 130 is a chromatogram of SEC analysis of AB1424 / 1612 F4 TriNKET after forced oxidation compared to control.

[0180] FIG. 131 is a graph showing reduced CE-SDS analysis of AB1424 / 1612 F4 TriNKET after forced oxidation compared to control.

[0181] FIG. 132 is a graph showing binding of AB1424 / 1612 F4 TriNKET to hBAFF-R+ cells after forced oxidation.

[0182] FIG. 133A and FIG. 133B are sensorgram graphs showing SPR binding of hCD16aV to AB1424 / 1612 F4 TriNKET control (FIG. 133A) and after forced oxidation (FIG. 133B).

[0183] FIG. 134 is a graph of a KHYG-l-CD16aV cytotoxicity assay of BJAB cells as induced by AB 1424 / 1612 F4 TriNKET after forced oxidation (red) and control (blue).

[0184] FIG. 135 is a chromatogram of SEC analysis of AB 1424 / 1612 F4 TriNKET after long term low pH stress compared to control.

[0185] FIG. 136 is a graph showing reduced CE-SDS analysis of AB1424 / 1612 F4 TriNKET after long term low pH stress compared to control.

[0186] FIG. 137 is a graph showing cIEF profiling of AB1424 / 1612 F4 TriNKET after long term low pH stress compared to control.

[0187] FIG. 138 is a graph showing binding of AB1424 / 1612 F4 TriNKET to hBAFF-R+ cells after long term low pH stress compared to control.

[0188] FIG. 139A and FIG. 139B are sensorgram graphs showing SPR binding of hCD16aV to AB 1424 / 1612 F4 TriNKET after long term low pH stress (FIG. 139B) compared to control (FIG. 139A).

[0189] FIG. 140 is a graph of a KHYG-l-CD16aV cytotoxicity assay of BJAB cells as induced by AB 1424 / 1612 F4 TriNKET after long term low pH stress (red) and control (blue).

[0190] FIG. 141 is a chromatogram of SEC analysis of AB 1424 / 1612 F4 TriNKET after long term high pH stress compared to control.

[0191] FIG. 142 is a graph showing reduced CE-SDS analysis of AB1424 / 1612 F4 TriNKET after long term high pH stress compared to control.

[0192] FIG. 143 is a graph showing cIEF profiling of AB1424 / 1612 F4 TriNKET after long term high pH stress compared to control.

[0193] FIG. 144 is a graph showing binding of AB1424 / 1612 F4 TriNKET to hBAFF-R+ cells after long term high pH stress (red) compared to control (blue).

[0194] FIG. 145 A and FIG. 145B are sensorgram graphs showing SPR binding of hCD16aV to AB 1424 / 1612 F4 TriNKET after long term high pH stress (FIG. 145B) compared to control (FIG. 145A).

[0195] FIG. 146 is a graph of a KHYG-l-CD16aV cytotoxicity assay of BJAB cells as induced by AB 1424 / 1612 F4 TriNKET after long term high pH stress (red) and control (blue).

[0196] FIG. 147 is a chromatogram of SEC analysis of AB1424 / 1612 F4 TriNKET after 6 freeze / thaw cycles compared to control.

[0197] FIG. 148 is a graph showing reduced CE-SDS analysis of AB1424 / 1612 F4 TriNKET after 6 freeze / thaw cycles compared to control.

[0198] FIG. 149 is a graph showing binding of AB1424 / 1612 F4 TriNKET to hBAFF-R+ cells after 6 freeze / thaw cycles (red) compared to control (blue).

[0199] FIG. 150 is a graph of a KHYG-l-CD16aV cytotoxicity assay of BJAB cells as induced by AB 1424 / 1612 F4 TriNKET after 6 freeze / thaw cycles (red) and control (blue).

[0200] FIG. 151 is a chromatogram of SEC analysis of AB 1424 / 1612 F4 TriNKET after agitation stress compared to control.

[0201] FIG. 152 is a graph showing reduced CE-SDS analysis of AB1424 / 1612 F4 TriNKET after agitation stress compared to control.

[0202] FIG. 153 is a graph showing binding of AB1424 / 1612 F4 TriNKET to hBAFF-R+ cells after agitation stress (red) compared to control (blue).

[0203] FIG. 154 is a graph of a KHYG-l-CD16aV cytotoxicity assay of BJAB cells as induced by AB 1424 / 1612 F4 TriNKET after agitation stress (red) and control (blue).

[0204] FIG. 155A and FIG. 155B is a chromatogram of SEC analysis of AB1424 / 1612 F4 TriNKET Protein A eluate pre- (FIG. 155 A) and post- low pH hold (FIG. 155B).

[0205] FIG. 156 is a graph showing cIEF profiling of AB1424 / 1612 F4 TriNKET after low pH hold compared to control.

[0206] FIG. 157 is a graph showing reduced CE-SDS analysis of AB1424 / 1612 F4 TriNKET after low pH hold compared to control.

[0207] FIG. 158 is a graph showing binding of AB1424 / 1612 F4 TriNKET to hBAFF-R+ cells after low pH hold (blue) compared to control (red).

[0208] FIG. 159 is a graph of a KHYG-l-CD16aV cytotoxicity assay of BJAB cells as induced by AB 1424 / 1612 F4 TriNKET after low pH hold (green) and control (red).

[0209] FIG. 160A and FIG. 160B are graphs showing binding of AB1424 / 1612 F3’ TriNKET (blue), AB 1424 / 1612 F4 TriNKET (red), and parental mAb (black) to KHYG-1 (FIG. 160A) and KHYG-1 -CD 16V (FIG. 160B) cell lines.

[0210] FIG. 161A and FIG. 161B are graphs showing percent surface retention of BAFF-R on RL cells exposed to AB1424 / 1612 F3’ TriNKET (blue), AB1424 / 1612 F4 TriNKET (red), and parental mAb (black) (FIG. 161 A) and activated with IL-2 (FIG. 16 IB).

[0211] FIG. 162 is a graph showing percent surface retention of BAFF-R on Raji cells exposed to AB1424 / 1612 F3’ TriNKET (blue), AB1424 / 1612 F4 TriNKET (red), and parental mAh (black).

[0212] FIG. 163 is a graph of a resting human NK cell-induced cytotoxicity assay of RL cells following incubation with AB 1424 / 1612 F3’ TriNKET (blue), AB1424 / 1612 F4 TriNKET (red), parental mAb (black), and human IgGlk (grey).

[0213] FIG. 164A and FIG. 164B are graphs of a rested human NK cell-induced cytotoxicity assay of RL cells following incubation with AB1424 / 1612 F3’ TriNKET (blue), AB1424 / 1612 F4 TriNKET (red), F3’ control (black), and F4 control (grey). Cells were cocultured with control (FIG. 164A) or IL-2 (FIG. 164B).

[0214] FIG. 165 is a graph of a KHYG-l-CD16aV cytotoxicity assay of BJAB cells as induced by AB1424 / 1612 F3’ TriNKET (blue), AB1424 / 1612 F3’ TriNKET lacking NKG2D binding (black), or AB1424 / 1612 F3’ TriNKET- Fc silenced (red), or palivizumab F3’ TriNKET (grey).

[0215] FIG. 166 is a graph of a resting human NK cell-induced cytotoxicity assay of BJAB cells as induced by AB1424 / 1612 F3’ TriNKET (blue), AB1424 / 1612 F3’ TriNKET lacking NKG2D binding (black), or AB 1424 / 1612 F3’ TriNKET- Fc silenced (red), or palivizumab F3’ TriNKET (grey).

[0216] FIG. 167 is a graph of a resting human NK cell-induced cytotoxicity assay of RL cells following incubation with AB 1424 / 1612 F3’ TriNKET (blue), AB1424 / 1612 F4 TriNKET (red), AB1424 / 1612 F3’ TriNKET plus soluble MICA (black), and AB1424 / 1612 F4 TriNKET plus soluble MICA (grey).

[0217] FIG. 168 is a graph of a resting human NK cell-induced cytotoxicity assay of RL cells following incubation with AB 1424 / 1612 F3’ TriNKET (blue), AB1424 / 1612 F4 TriNKET (red), AB 1424 / 1612 F3’ TriNKET plus BAFF (black), and AB 1424 / 1612 F4 TriNKET plus BAFF (grey).

[0218] FIG. 169 is a graph of interferon gamma (IFNy) and CD 107a production by BJAB cells following incubation with AB 1424 / 1612 F3’ TriNKET (blue), AB1424 / 1612 F4 TriNKET (red), parental mAb (black), F3’-palivisumab (light grey), and F4-palivisumab (dark grey).

[0219] FIG. 170 is a graph of phagocytosis of B JAB cells by M0 macrophages following incubation with AB1424 / 1612 F3’ TriNKET (blue), AB1424 / 1612 F4 TriNKET (red), parental mAb (black), and Fc-silenced AB1424 / 1612 F3’ TriNKET (pink).

[0220] FIG. 171 is a graph of a human serum-induced cytotoxicity assay of Raji cells following incubation with rituximab (black), AB1424 / 1612 F3’ TriNKET (blue), or AB1424 / 1612 F3’ TriNKET.

[0221] FIG. 172A-FIG. 172E are histograms showing flow cytometry analysis of binding of AB1424 / 1612 F3’ TriNKET (blue) and F3’-palivizumab (red) to indicated BAFF-R+ cells in PBMCs.

[0222] FIG. 173A-FIG. 173F are histograms showing flow cytometry analysis of binding of AB1424 / 1612 F3’ TriNKET (blue) and F3’-palivizumab (red) to indicated cell types in human blood.

[0223] FIG. 174A-FIG. 174C are histograms showing flow cytometry analysis of binding of AB1424 / 1612 F3’ TriNKET (blue) and F3’-palivizumab (red) to human red blood cells.

[0224] FIG. 175A-FIG. 175F are graphs showing flow cytometry analysis of binding of (from left to right) AB1424 / 1612 F3’ TriNKET, F3’-palivizumab, AB1424 / 1612 F4 TriNKET, F4-palivizumab, and rituximab to indicated human donor PBMCs.

[0225] FIG. 176A-FIG. 176F are histograms showing flow cytometry analysis of binding of AB1424 / 1612 F3’ TriNKET (blue) and F3’-palivizumab (red) to indicated PBMCs from cynomolgus whole blood donor CYN317060.

[0226] FIG. 177A-FIG. 177F are graphs showing flow cytometry analysis of binding of (from left to right) AB1424 / 1612 F3’ TriNKET, F3’-palivizumab, AB1424 / 1612 F4 TriNKET, F4-palivizumab, and rituximab to indicated human donor PBMCs.

[0227] FIG. 178 is a graph showing CD 107a positivity of CD 16+ CD8+ NK cells in a coculture of BJAB cells with PBMCs from cynomolgus whole blood donor CYN317060.DETAILED DESCRIPTION

[0228] The present application provides multispecific binding proteins that bind the NKG2D receptor and CD 16 receptor on natural killer cells, and BAFF-R on a cancer cell or a Bcell. In some embodiments, the multispecific proteins further include an additional antigenbinding site that binds BAFF-R. The application also provides pharmaceutical compositions comprising such multispecific binding proteins, and therapeutic methods using such multispecific proteins and pharmaceutical compositions, for purposes such as treating autoimmune diseases and cancer. Various aspects of the multispecific binding proteins described in the present application are set forth below in sections; however, aspects of the multispecific binding proteins described in one particular section are not to be limited to any particular section.

[0229] To facilitate an understanding of the present application, a number of terms and phrases are defined below.

[0230] The terms “a” and “an” as used herein mean “one or more” and include the plural unless the context is inappropriate.

[0231] As used herein, the term “antigen-binding site” refers to the part of the immunoglobulin molecule that participates in antigen binding. In human antibodies, the antigen binding site is formed by amino acid residues of the N-terminal variable (“V”) regions of the heavy (“H”) and light (“L”) chains. Three highly divergent stretches within the V regions of the heavy and light chains are referred to as “hypervariable regions” which are interposed between more conserved flanking stretches known as “framework regions,” or “FR .” Thus the term “FR” refers to amino acid sequences which are naturally found between and adjacent to hypervariable regions in immunoglobulins. In a human antibody molecule, the three hypervariable regions of a light chain and the three hypervariable regions of a heavy chain are disposed relative to each other in three-dimensional space to form an antigen-binding surface. The antigen-binding surface is complementary to the three-dimensional surface of a bound antigen, and the three hypervariable regions of each of the heavy and light chains are referred to as “complementarity-determining regions,” or “CDRs.” In certain animals, such as camels and cartilaginous fish, the antigen-binding site is formed by a single antibody chain providing a “single domain antibody.” Antigen-binding sites can exist in an intact antibody, in an antigen-binding fragment of an antibody that retains the antigen-binding surface, or in a recombinant polypeptide such as an scFv, using a peptide linker to connect the heavy chain variable domain to the light chain variable domain in a single polypeptide.

[0232] The term “tumor-associated antigen” as used herein means any antigen including but not limited to a protein, glycoprotein, ganglioside, carbohydrate, or lipid that is associated with cancer. Such antigen can be expressed on malignant cells or in the tumor microenvironment such as on tumor-associated blood vessels, extracellular matrix, mesenchymal stroma, or immune infiltrates. In certain embodiments of the present disclosure, the terms “tumor- associated antigen” refers to BAFF-R, which is targeted by the second and / or the additional antigen-binding site present in a multispecific binding proteins of the present disclosure. It is understood, however, that BAFF-R may also be associated with diseases and disorders that are not tumor or cancer.

[0233] As used herein, the terms “subject” and “patient” refer to an organism to be treated by the methods and compositions described herein. Such organisms preferably include, but are not limited to, mammals (e.g., murines, simians, equines, bovines, porcines, canines, felines, and the like), and more preferably include humans.

[0234] As used herein, the term “effective amount” refers to the amount of a compound (e.g., a compound of the present application) sufficient to effect beneficial or desired results. An effective amount can be administered in one or more administrations, applications or dosages and is not intended to be limited to a particular formulation or administration route. As used herein, the term “treating” includes any effect, e.g., lessening, reducing, modulating, ameliorating or eliminating, that results in the improvement of the condition, disease, disorder, and the like, or ameliorating a symptom thereof.

[0235] As used herein, the term “pharmaceutical composition” refers to the combination of an active agent with a carrier, inert or active, making the composition especially suitable for diagnostic or therapeutic use in vivo or ex vivo.

[0236] As used herein, the term “pharmaceutically acceptable carrier” refers to any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, emulsions (e.g., such as an oil / water or water / oil emulsions), and various types of wetting agents. The compositions also can include stabilizers and preservatives. For examples of carriers, stabilizers and adjuvants, see e.g., Martin, Remington's Pharmaceutical Sciences, 15th Ed., Mack Publ. Co., Easton, PA

[1975] ,

[0237] As used herein, the term “pharmaceutically acceptable salt” refers to any pharmaceutically acceptable salt (e.g., acid or base) of a compound described in the present application which, upon administration to a subject, is capable of providing a compound described in this application or an active metabolite or residue thereof. As is known to those of skill in the art, “salts” of the compounds described in the present application may be derived from inorganic or organic acids and bases. Exemplary acids include, but are not limited to, hydrochloric, hydrobromic, sulfuric, nitric, perchloric, fumaric, maleic, phosphoric, glycolic, lactic, salicylic, succinic, toluene-p-sulfonic, tartaric, acetic, citric, methanesulfonic, ethanesulfonic, formic, benzoic, malonic, naphthalene-2-sulfonic, benzenesulfonic acid, and the like. Other acids, such as oxalic, though not in themselves pharmaceutically acceptable, may be employed in the preparation of salts useful as intermediates in obtaining the compounds described in the application and their pharmaceutically acceptable acid addition salts.

[0238] Exemplary bases include, but are not limited to, alkali metal (e.g., sodium) hydroxides, alkaline earth metal (e.g., magnesium) hydroxides, ammonia, and compounds of formula NWf, wherein W is Ci-4 alkyl, and the like.

[0239] Exemplary salts include, but are not limited to: acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, flucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2- naphthalenesulfonate, nicotinate, oxalate, palmoate, pectinate, persulfate, phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, tosylate, undecanoate, and the like. Other examples of salts include anions of the compounds described in the present application compounded with a suitable cation such as Na+, NH4+, and NW4+(wherein W is a Ci-4 alkyl group), and the like.

[0240] For therapeutic use, salts of the compounds described in the present application are contemplated as being pharmaceutically acceptable. However, salts of acids and bases that are non-pharmaceutically acceptable may also find use, for example, in the preparation or purification of a pharmaceutically acceptable compound.

[0241] As used herein, BAFF-R (also known as BAFF receptor, B-cell activating factorreceptor, BR3, TNFRSF13C, tumor necrosis factor receptor superfamily member 13C, TNF receptor superfamily member 13C, CD268, and BLyS receptor 3) refers to the protein of Uniprot Accession No. Q96RJ3 and related isoforms and orthologs.

[0242] Throughout the description, where compositions are described as having, including, or comprising specific components, or where processes and methods are described as having, including, or comprising specific steps, it is contemplated that, additionally, there are compositions described in the present application that consist essentially of, or consist of, the recited components, and that there are processes and methods according to the present application that consist essentially of, or consist of, the recited processing steps.

[0243] As a general matter, compositions specifying a percentage are by weight unless otherwise specified. Further, if a variable is not accompanied by a definition, then the previous definition of the variable controls.I. PROTEINS

[0244] The present application provides multispecific binding proteins that bind to the NKG2D receptor and CD16 receptor on natural killer cells, and BAFF-R on a cancer cell. The multispecific binding proteins are useful in the pharmaceutical compositions and therapeutic methods described herein. Binding of the multispecific binding proteins to the NKG2D receptor and CD 16 receptor on a natural killer cell enhances the activity of the natural killer cell toward destruction of tumor cells expressing BAFF-R antigen. Binding of the multispecific binding proteins to BAFF-R-expressing cells brings the cancer cells into proximity with the natural killer cell, which facilitates direct and indirect destruction of the tumor cells by the natural killer cell. Multispecific binding proteins that bind NKG2D, CD 16, and another target are disclosed in International Application Publication Nos. WO2018148445 and WO2019157366, which are not incorporated herein by reference. Further description of some exemplary multispecific binding proteins is provided below.

[0245] The first component of the multispecific binding protein is an antigen-binding site that binds to NKG2D receptor-expressing cells, which can include but are not limited to NK cells, y6 T cells and CD8+aP T cells. Upon NKG2D binding, the multispecific binding proteins may block natural ligands, such as ULBP6 and MICA, from binding to NKG2D and activating NK cells.

[0246] The second component of the multispecific binding protein is an antigen-binding site that binds to BAFF-R. The BAFF-R-expressing cells may be found, for example, in B-cell non-Hodgkin’s lymphoma (B-NHL), such as chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), marginal zone lymphoma, mucosa-associated lymphoid tissue (MALT) lymphoma, primary mediastinal B-cell lymphoma, acute lymphocytic leukemia (ALL); and autoimmune inflammatory diseases.

[0247] The third component of the multispecific binding proteins is an antibody Fc domain or a portion thereof, or an antigen-binding site that binds to cells expressing CD16, an Fc receptor on the surface of leukocytes including natural killer cells, macrophages, neutrophils, eosinophils, mast cells, and follicular dendritic cells.

[0248] An additional antigen-binding site of the multispecific binding proteins may bind BAFF-R. In certain embodiments, the first antigen-binding site that binds NKG2D is an scFv, and the second and the additional antigen-binding sites that bind BAFF-R are each a Fab fragment. In certain embodiments, the first antigen-binding site that binds NKG2D is an scFv, and the second and the additional antigen-binding sites that bind BAFF-R are each an scFv. In certain embodiments, the first antigen-binding site that binds NKG2D is a Fab fragment, and the second and the additional antigen-binding sites that bind BAFF-R are each an scFv. In certain embodiments, the first antigen-binding site that binds NKG2D is a Fab, and the second and the additional antigen-binding sites that bind BAFF-R are each a Fab fragment.

[0249] The multispecific binding proteins described herein can take various formats. For example, one format is a heterodimeric, multispecific antibody including a first immunoglobulin heavy chain, a first immunoglobulin light chain, a second immunoglobulin heavy chain and a second immunoglobulin light chain (FIG. 1). The first immunoglobulin heavy chain includes a first Fc (hinge-CH2-CH3) domain, a first heavy chain variable domain and optionally a first CHI heavy chain domain. The first immunoglobulin light chain includes a first light chain variable domain and optionally a first light chain constant domain. The first immunoglobulin light chain, together with the first immunoglobulin heavy chain, forms an antigen-binding site that binds NKG2D. The second immunoglobulin heavy chain comprises a second Fc (hinge-CH2-CH3) domain, a second heavy chain variable domain and optionally a second CHI heavy chaindomain. The second immunoglobulin light chain includes a second light chain variable domain and optionally a second light chain constant domain. The second immunoglobulin light chain, together with the second immunoglobulin heavy chain, forms an antigen-binding site that binds BAFF-R. In some embodiments, the first Fc domain and second Fc domain together are able to bind to CD 16 (FIG. 1). In some embodiments, the first immunoglobulin light chain is identical to the second immunoglobulin light chain.

[0250] The antigen-binding sites may each incorporate an antibody heavy chain variable domain and an antibody light chain variable domain (e.g., arranged as in an antibody, or fused together to form an scFv), or one or more of the antigen-binding sites may be a single domain antibody, such as a VHH antibody like a camelid antibody or a VNAR antibody like those found in cartilaginous fish.

[0251] In some embodiments, the second antigen-binding site incorporates a light chain variable domain having an amino acid sequence identical to the amino acid sequence of the light chain variable domain present in the first antigen-binding site.

[0252] Another exemplary format involves a heterodimeric, multispecific antibody including a first immunoglobulin heavy chain, a second immunoglobulin heavy chain and an immunoglobulin light chain (e.g., FIG. 2A). In some embodiments, the first immunoglobulin heavy chain includes a first Fc (hinge-CH2-CH3) domain fused via either a linker or an antibody hinge to a single-chain variable fragment (scFv) composed of a heavy chain variable domain and light chain variable domain which pair and bind NKG2D, or bind BAFF-R. In some embodiments, the second immunoglobulin heavy chain includes a second Fc (hinge-CH2-CH3) domain, a second heavy chain variable domain and a CHI heavy chain domain. The immunoglobulin light chain includes a light chain variable domain and a light chain constant domain. In some embodiments, the second immunoglobulin heavy chain pairs with the immunoglobulin light chain and binds to NKG2D or binds BAFF-R with the proviso that when the first Fc domain is fused to an scFv that binds NKG2D, the second immunoglobulin heavy chain paired with the immunoglobulin light chain binds BAFF-R but not NKG2D, and vice versa. In some embodiments, the scFv in the first immunoglobulin heavy chain binds BAFF-R; and the heavy chain variable domain in the second immunoglobulin heavy chain and the light chain variable domain in the immunoglobulin light chain, when paired, bind NKG2D (e.g., FIG.2E). In some embodiments, the scFv in the first immunoglobulin heavy chain binds NKG2D; and the heavy chain variable domain in the second immunoglobulin heavy chain and the light chain variable domain in the immunoglobulin light chain, when paired, bind BAFF-R. In some embodiments, the first Fc domain and the second Fc domain together are able to bind to CD 16 (e.g., FIG. 2A). In some embodiments, the first Fc domain and the second Fc domain together are able to bind to CD 16 (e.g., FIG. 2A).

[0253] Another exemplary format involves a heterodimeric, multispecific antibody including a first immunoglobulin heavy chain, and a second immunoglobulin heavy chain (e.g., FIG. 2B). In some embodiments, the first immunoglobulin heavy chain includes a first Fc (hinge-CH2-CH3) domain fused via either a linker or an antibody hinge to a single-chain variable fragment (scFv) composed of a heavy chain variable domain and light chain variable domain, which pair and bind NKG2D, or bind BAFF-R. In some embodiments, the second immunoglobulin heavy chain includes a second Fc (hinge-CH2-CH3) domain fused via either a linker or an antibody hinge to a single-chain variable fragment (scFv) composed of a heavy chain variable domain and light chain variable domain which pair and bind NKG2D or bind BAFF-R, with the proviso that when the first Fc domain is fused to an scFv that binds NKG2D, the second Fc domain fused to an scFv binds BAFF-R, but not NKG2D, and vice versa. In some embodiments, the first Fc domain and the second Fc domain together are able to bind to CD 16 (e.g., FIG. 2B)

[0254] In some embodiments, the single-chain variable fragment (scFv) described above is linked to the antibody constant domain via a hinge sequence. In some embodiments, the hinge comprises amino acids Ala-Ser or Gly-Ser. In some embodiments, the hinge comprises amino acids Ala-Ser or Gly-Ser. In some embodiments, the hinge connecting an scFv (e.g., an scFv that binds NKG2D or an scFv that binds BAFF-R) and the antibody heavy chain constant domain comprises amino acids Ala-Ser. In some embodiments, the hinge connecting an scFv (e.g., an scFv that binds NKG2D or an scFv that binds BAFF-R) and the antibody heavy chain constant domain comprises amino acids Gly-Ser. In some other embodiments, the hinge comprises amino acids Ala-Ser and Thr-Lys-Gly. The hinge sequence can provide flexibility of binding to the target antigen, and balance between flexibility and optimal geometry.

[0255] In some embodiments, the single-chain variable fragment (scFv) described above includes a heavy chain variable domain and a light chain variable domain. In some embodiments, the heavy chain variable domain forms a disulfide bridge with the light chain variable domain to enhance stability of the scFv. For example, a disulfide bridge can be formed between the C44 residue of the heavy chain variable domain and the Cl 00 residue of the light chain variable domain, the amino acid positions numbered under Kabat. In some embodiments, the heavy chain variable domain is linked to the light chain variable domain via a flexible linker. Any suitable linker can be used, for example, the (G4S)4 linker ((GlyGlyGlyGlySer)4 (SEQ ID NO: 119)). In some embodiments of the scFv, the heavy chain variable domain is positioned at the N-terminus of the light chain variable domain. In some embodiments of the scFv, the heavy chain variable domain is positioned at the C terminus of the light chain variable domain.

[0256] The multispecific binding proteins described herein can further include one or more additional antigen-binding sites. The additional antigen-binding site(s) may be fused to the N- terminus of the constant region CH2 domain or to the C-terminus of the constant region CH3 domain, optionally via a linker sequence. In certain embodiments, the additional antigen-binding site(s) takes the form of a single-chain variable region (scFv) that is optionally disulfide- stabilized, resulting in a tetravalent or trivalent multispecific binding protein. For example, a multispecific binding protein includes a first antigen-binding site that binds NKG2D, a second antigen-binding site that binds BAFF-R, an additional antigen-binding site that binds BAFF-R, and an antibody constant region or a portion thereof sufficient to bind CD 16 or a fourth antigenbinding site that binds CD 16. Any one of these antigen binding sites can either take the form of a Fab fragment or an scFv, such as an scFv described above.

[0257] In some embodiments, the additional antigen-binding site binds a different epitope of BAFF-R from the second antigen-binding site. In some embodiments, the additional antigenbinding site binds the same epitope as the second antigen-binding site. In some embodiments, the additional antigen-binding site comprises the same heavy chain and light chain CDR sequences as the second antigen-binding site. In some embodiments, the additional antigen-binding site comprises the same heavy chain and light chain variable domain sequences as the second antigen-binding site. In some embodiments, the additional antigen-binding site has the same amino acid sequence(s) as the second antigen-binding site. In some embodiments, the additional antigen-binding site comprises heavy chain and light chain variable domain sequences that aredifferent from the heavy chain and light chain variable domain sequences of the second antigenbinding site. In some embodiments, the additional antigen-binding site has an amino acid sequence that is different from the sequence of the second antigen-binding site. In some embodiments, the second antigen-binding site and the additional antigen-binding site bind different tumor-associated antigens. In some embodiments, the second antigen-binding site and the additional antigen-binding site binds different antigens. Exemplary formats are shown in FIG. 2C and FIG. 2D. Accordingly, the multispecific binding proteins can provide bivalent engagement of BAFF-R. Bivalent engagement of BAFF-R by the multispecific proteins can stabilize BAFF-R on the tumor cell surface and enhance cytotoxicity of NK cells towards the tumor cells. Bivalent engagement of BAFF-R by the multispecific proteins can confer stronger binding of the multispecific proteins to the tumor cells, thereby facilitating stronger cytotoxic response of NK cells towards the tumor cells, especially towards tumor cells expressing a low level of BAFF-R.

[0258] The multispecific binding proteins can take additional formats. In some embodiments, the multispecific binding protein is in the Triomab form, which is a trifunctional, bispecific antibody that maintains an IgG-like shape. This chimera consists of two half antibodies, each with one light and one heavy chain, that originate from two parental antibodies.

[0259] In some embodiments, the multispecific binding protein is in a KiH Common Light Chain (LC) form, which incorporates the knobs-into-holes (KiH) technology (e.g., the multispecific binding protein represented in FIG. 21). The KiH Common LC form is a heterodimer comprising a Fab which binds to a first target, a Fab which binds to a second target, and an Fc domain stabilized by heterodimerization mutations. The two Fabs each comprise a heavy chain and light chain, wherein the heavy chain of each Fab differs from the other, and the light chain that pairs with each respective heavy chain is common to both Fabs.

[0260] In some embodiments, the multispecific binding protein is the KiH form, which involves the knobs-into-holes (KiHs) technology. The KiH involves engineering CH3 domains to create either a “knob” or a “hole” in each heavy chain to promote heterodimerization. The concept behind the “Knobs-into-Holes (KiH)” Fc technology was to introduce a “knob” in one CH3 domain (CH3A) by substitution of a small residue with a bulky one (e.g., T366WCH3A in EU numbering). To accommodate the “knob,” a complementary “hole” surface was created onthe other CH3 domain (CH3B) by replacing the closest neighboring residues to the knob with smaller ones (e.g., T366S / L368A / Y407VCH3B). The “hole” mutation was optimized by structured-guided phage library screening (Atwell S, Ridgway JB, Wells JA, Carter P., Stable heterodimers from remodeling the domain interface of a homodimer using a phage display library, J. Mol. Biol. (1997) 270(l):26-35). X-ray crystal structures of KiH Fc variants (Elliott JM, Ultsch M, Lee J, Tong R, Takeda K, Spiess C, et al., Antiparallel conformation of knob and hole aglycosylated half-antibody homodimers is mediated by a CH2-CH3 hydrophobic interaction. J. Mol. Biol. (2014) 426(9): 1947-57; Mimoto F, Kadono S, Katada H, Igawa T, Kamikawa T, Hattori K. Crystal structure of a novel asymmetrically engineered Fc variant with improved affinity for FcyRs. Mol. Immunol. (2014) 58(1): 132-8) demonstrated that heterodimerization is thermodynamically favored by hydrophobic interactions driven by steric complementarity at the inter-CH3 domain core interface, whereas the knob-knob and the holehole interfaces do not favor homodimerization owing to steric hindrance and disruption of the favorable interactions, respectively.

[0261] In some embodiments, the multispecific binding protein is in the dual-variable domain immunoglobulin (DVD-Ig™) form, which combines the target binding domains of two monoclonal antibodies via flexible naturally occurring linkers, and yields a tetravalent IgG-like molecule.

[0262] In some embodiments, the multispecific binding protein is in the Orthogonal Fab interface (Ortho-Fab) form. In the ortho-Fab IgG approach (Lewis SM, Wu X, Pustilnik A, Sereno A, Huang F, Rick HL, et al., Generation of bispecific IgG antibodies by structure-based design of an orthogonal Fab interface. Nat. Biotechnol. (2014) 32(2): 191-8), structure-based regional design introduces complementary mutations at the LC and HCVH-CHI interface in only one Fab fragment, without any changes being made to the other Fab fragment.

[0263] In some embodiments, the multispecific binding protein is in the 2-in-l Ig format. In some embodiments, the multispecific binding protein is in the ES form, which is a heterodimeric construct containing two different Fab fragments binding to targets 1 and target 2 fused to the Fc. Heterodimerization is ensured by electrostatic steering mutations in the Fc.

[0264] In some embodiments, the multispecific binding protein is in the i<k-Body form, which is a heterodimeric construct with two different Fab fragments fused to Fc stabilized byheterodimerization mutations: Fab fragment 1 targeting antigen 1 contains kappa LC, and Fab fragment 2 targeting antigen 2 contains lambda LC. FIG. 13A is an exemplary representation of one form of a i<k-Body; FIG. 13B is an exemplary representation of another i<A-Body.

[0265] In some embodiments, the multispecific binding protein is in Fab Arm Exchange form (antibodies that exchange Fab fragment arms by swapping a heavy chain and attached light chain (half-molecule) with a heavy -light chain pair from another molecule, which results in bispecific antibodies).

[0266] In some embodiments, the multispecific binding protein is in the SEED Body form. The strand-exchange engineered domain (SEED) platform was designed to generate asymmetric and bispecific antibody-like molecules, a capability that expands therapeutic applications of natural antibodies. This protein engineering platform is based on exchanging structurally related sequences of immunoglobulin within the conserved CH3 domains. The SEED design allows efficient generation of AG / GA heterodimers, whereas disfavoring homodimerization of AG and GA SEED CH3 domains. (Muda M. et al., Protein Eng. Des. Sei. (2011, 24(5):447-54)).

[0267] In some embodiments, the multispecific binding protein is in the LuZ-Y form, in which a leucine zipper is used to induce heterodimerization of two different HCs. (Wranik, BJ. et al., J. Biol. Chem. (2012), 287:43331-9).

[0268] In some embodiments, the multispecific binding protein is in the Cov-X-Body form. In bispecific CovX-Bodies, two different peptides are joined together using a branched azetidinone linker and fused to the scaffold antibody under mild conditions in a site-specific manner. Whereas the pharmacophores are responsible for functional activities, the antibody scaffold imparts long half-life and Ig-like distribution. The pharmacophores can be chemically optimized or replaced with other pharmacophores to generate optimized or unique bispecific antibodies. (Doppalapudi VR et al., PNAS (2010), 107(52);22611-22616).

[0269] In some embodiments, the multispecific binding protein is in an OAsc-Fab heterodimeric form that includes Fab fragment binding to target 1, and scFab binding to target 2 fused to Fc. Heterodimerization is ensured by mutations in the Fc.

[0270] In some embodiments, the multispecific binding protein is in a DuetMab form, which is a heterodimeric construct containing two different Fab fragments binding to antigens 1and 2, and Fc stabilized by heterodimerization mutations. Fab fragments 1 and 2 contain differential S-S bridges that ensure correct LC and HC pairing.

[0271] In some embodiments, the multispecific binding protein is in a CrossmAb form, which is a heterodimeric construct with two different Fab fragments binding to targets 1 and 2, fused to Fc stabilized by heterodimerization. CL and CHI domains and VH and VL domains are switched, e.g., CHI is fused in-frame with VL, and CL is fused in-frame with VH.

[0272] In some embodiments, the multispecific binding protein is in a Fit-Ig form, which is a homodimeric construct where Fab fragment binding to antigen 2 is fused to the N terminus of HC of Fab fragment that binds to antigen 1. The construct contains wild-type Fc.

[0273] Individual components of the multispecific binding proteins are described in more detail below.NKG2D-Binding Site

[0274] Upon binding to the NKG2D receptor and CD 16 receptor on natural killer cells, and BAFF-R, the multispecific binding proteins can engage more than one kind of NK-activating receptor, and may block the binding of natural ligands to NKG2D. In certain embodiments, the proteins can agonize NK cells in humans. In some embodiments, the proteins can agonize NK cells in humans and in other species such as rodents and cynomolgus monkeys. In some embodiments, the proteins can agonize NK cells in humans and in other species such as cynomolgus monkeys.

[0275] Table 1 lists peptide sequences of heavy chain variable domains and light chain variable domains that, in combination, can bind to NKG2D. In some embodiments, the heavy chain variable domain and the light chain variable domain are arranged in Fab format. In some embodiments, the heavy chain variable domain and the light chain variable domain are fused together to form an scFv.

[0276] The NKG2D binding sites listed in Table 1 can vary in their binding affinity to NKG2D, nevertheless, they all activate human NK cells.

[0277] Unless indicated otherwise, the CDR sequences provided in Table 1 are determined under Kabat numbering.

[0278] In certain embodiments, the first antigen-binding site that binds NKG2D (e.g., human NKG2D) comprises an antibody heavy chain variable domain (VH) that comprises an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the VH of an antibody disclosed in Table 1, and an antibody light chain variable domain (VL) that comprises an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the VL of the same antibody disclosed in Table 1. In certain embodiments, the first antigenbinding site comprises the heavy chain CDR1, CDR2, and CDR3 and the light chain CDR1, CDR2, and CDR3, determined under Kabat (see Kabat et al., (1991) Sequences of Proteins of Immunological Interest, NIH Publication No. 91-3242, Bethesda), Chothia (see, e.g., Chothia C & Lesk A M, (1987), J. Mol. Biol. 196: 901-917), MacCallum (see MacCallum R M et al., (1996) J. Mol. Biol. 262: 732-745), or any other CDR determination method known in the art, of the VH and VL sequences of an antibody discloses in Table 1. In certain embodiments, the first antigen-binding site comprises the heavy chain CDR1, CDR2, and CDR3 and the light chain CDR1, CDR2, and CDR3 of an antibody disclosed in Table 1.

[0279] In certain embodiments, the first antigen-binding site that binds to NKG2D comprises a heavy chain variable domain derived from SEQ ID NO: 1, such as by having an amino acid sequence at least 90% (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 1, and / or incorporating amino acid sequences identical to the CDR1 (SEQ ID NO:2), CDR2 (SEQ ID NO:3), and CDR3 (SEQ ID NO:4) sequences of SEQ ID NO: 1. The heavy chain variable domain related to SEQ ID NO: 1 can be coupled with a variety of light chain variable domains to form an NKG2D binding site. For example, the first antigen-binding site that incorporates a heavy chain variable domain related to SEQ ID NO: 1 can further incorporate a light chain variable domain selected from the sequences derived from SEQ ID NOs: 5, 6, 7, 8, 9, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, and 46. For example, the first antigen-binding site incorporates a heavy chain variable domain with amino acid sequences at least 90% (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 1 and a light chain variable domain with amino acid sequences at least 90% (e.g., at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to any one of the sequences selected from SEQ ID NOs: 5, 6, 7, 8, 9, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, and 46.

[0280] In certain embodiments, the first antigen-binding site that binds NKG2D comprises a VH that comprises an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:26, and a VL that comprises an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO:32. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 27 or 28, 29, and 30 or 31, respectively (e.g., SEQ ID NOs: 27, 29, and 30, respectively, or SEQ ID NOs: 28, 29, and 31, respectively). In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 33, 34, and 35, respectively. In certain embodiments, the first antigen-binding site comprises (a) a VH that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 27 or 28, 29, and 30 or 31, respectively e.g., SEQ ID NOs: 27, 29, and 30, respectively, or SEQ ID NOs: 28, 29, and 31, respectively); and (b) a VL that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 33, 34, and 35, respectively.

[0281] In certain embodiments, the first antigen-binding site that binds NKG2D comprises a VH that comprises an amino acid sequence at least 90% (e.g, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:36, and a VL that comprises an amino acid sequence at least 90% (e.g, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO:42. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 37 or 38, 39, and 40 or 41, respectively (e.g., SEQ ID NOs: 37, 39, and 40, respectively, or SEQ ID NOs: 38, 39, and 41, respectively). In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 43, 44, and 45, respectively. In certain embodiments, the first antigen-binding site comprises (a) a VH that comprises CDR1, CDR2, and CDR3 comprising the amino acidsequences of SEQ ID NOs: 37 or 38, 39, and 40 or 41, respectively (e.g., SEQ ID NOs: 37, 39, and 40, respectively, or SEQ ID NOs: 38, 39, and 41, respectively); and (b) a VL that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 43, 44, and 45, respectively.

[0282] In certain embodiments, the first antigen-binding site that binds NKG2D comprises a VH that comprises an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:47, and a VL that comprises an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO:49. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 27, 29, and 48, respectively. In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 50, 34, and 51, respectively. In certain embodiments, the first antigen-binding site comprises (a) a VH that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 27, 29, and 48, respectively; and (b) a VL that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 50, 34, and 51, respectively.

[0283] In certain embodiments, the first antigen-binding site that binds NKG2D comprises a VH that comprises an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:52, and a VL that comprises an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO:58. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 53 or 54, 55, and 56 or 57, respectively (e.g., SEQ ID NOs: 53, 55, and 56, respectively, or SEQ ID NOs: 54, 55, and 57, respectively). In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 59, 60, and 61, respectively. In certain embodiments, the first antigen-binding site comprises (a) a VH that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 53 or 54, 55, and 56 or 57, respectively (e.g., SEQ ID NOs: 53, 55, and 56, respectively, or SEQ ID NOs: 54, 55, and 57, respectively); and (b) a VL that comprisesCDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 59, 60, and 61, respectively.

[0284] In certain embodiments, the first antigen-binding site that binds NKG2D comprises a VH that comprises an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:62, and a VL that comprises an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO:68. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 63 or 64, 65, and 66 or 67, respectively (e.g., SEQ ID NOs: 63, 65, and 66, respectively, or SEQ ID NOs: 64, 65, and 67, respectively). In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 59, 60, and 69, respectively. In certain embodiments, the first antigen-binding site comprises (a) a VH that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 63 or 64, 65, and 66 or 67, respectively (e.g., SEQ ID NOs: 63, 65, and 66, respectively, or SEQ ID NOs: 64, 65, and 67, respectively); and (b) a VL that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 59, 60, and 69, respectively.

[0285] In certain embodiments, the first antigen-binding site that binds NKG2D comprises a VH that comprises an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:89, and a VL that comprises an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO:92. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 53 or 54, 55, and 90 or 91, respectively (e.g., SEQ ID NOs: 53, 55, and 90, respectively, or SEQ ID NOs: 54, 55, and 91, respectively). In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 93, 44, and 94, respectively. In certain embodiments, the first antigen-binding site comprises (a) a VH that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 53 or 54, 55, and 90 or 91, respectively (e.g., SEQ ID NOs: 53, 55,and 90, respectively, or SEQ ID NOs: 54, 55, and 91, respectively); and (b) a VL that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 93, 44, and 94, respectively.

[0286] In certain embodiments, the first antigen-binding site that binds NKG2D comprises a VH that comprises an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:70, and a VL that comprises an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO:75. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 71 or 115, 72, and 73 or 74, respectively (e.g., SEQ ID NOs: 71, 72, and 73, respectively, or SEQ ID NOs: 115, 72, and 74, respectively). In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 76, 77, and 78, respectively. In certain embodiments, the first antigen-binding site comprises (a) a VH that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 71 or 115, 72, and 73 or 74, respectively e.g., SEQ ID NOs: 71, 72, and 73, respectively, or SEQ ID NOs: 115, 72, and 74, respectively); and (b) a VL that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 76, 77, and 78, respectively.

[0287] In certain embodiments, the first antigen-binding site that binds NKG2D comprises a VH that comprises an amino acid sequence at least 90% (e.g, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:79, and a VL that comprises an amino acid sequence at least 90% (e.g, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO:85. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 80 or 81, 82, and 83 or 84, respectively (e.g., SEQ ID NOs: 80, 82, and 83, respectively, or SEQ ID NOs: 81, 82, and 84, respectively). In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 86, 77, and 87, respectively. In certain embodiments, the first antigen-binding site comprises (a) a VH that comprises CDR1, CDR2, and CDR3 comprising the amino acidsequences of SEQ ID NOs: 80 or 81, 82, and 83 or 84 respectively (e.g., SEQ ID NOs: 80, 82, and 83, respectively, or SEQ ID NOs: 81, 82, and 84, respectively); and (b) a VL that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 86, 77, and 87, respectively.

[0288] In certain embodiments, the first antigen-binding site that binds NKG2D comprises a VH that comprises an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:95, and a VL that comprises an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO:85. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 80 or 81, 82, and 96 or 97, respectively (e.g., SEQ ID NOs: 80, 82, and 96, respectively, or SEQ ID NOs: 81, 82, and 97, respectively). In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 86, 77, and 87, respectively. In certain embodiments, the first antigen-binding site comprises (a) a VH that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 80 or 81, 82, and 96 or 97, respectively (e.g., SEQ ID NOs: 80, 82, and 96, respectively, or SEQ ID NOs: 81, 82, and 97, respectively); and (b) a VL that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 86, 77, and 87, respectively.

[0289] In certain embodiments, the first antigen-binding site that binds NKG2D comprises a VH that comprises an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:98, and a VL that comprises an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO:85. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 80 or 81, 82, and 99 or 100, respectively (e.g., SEQ ID NOs: 80, 82, and 99, respectively, or SEQ ID NOs: 81, 82, and 100, respectively). In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 86, 77, and 87, respectively. In certain embodiments, the first antigen-bindingsite comprises (a) a VH that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 80 or 81, 82, and 99 or 100, respectively (e.g., SEQ ID NOs: 80, 82, and 99, respectively, or SEQ ID NOs: 81, 82, and 100, respectively); and (b) a VL that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 86, 77, and 87, respectively.

[0290] In certain embodiments, the first antigen-binding site that binds NKG2D comprises a VH that comprises an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 101, and a VL that comprises an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO:85. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 80 or 81, 82, and 102 or 103, respectively (e.g., SEQ ID NOs: 80, 82, and 102, respectively, or SEQ ID NOs: 81, 82, and 103, respectively). In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 86, 77, and 87, respectively. In certain embodiments, the first antigen-binding site comprises (a) a VH that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 80 or 81, 82, and 102 or 103, respectively (e.g., SEQ ID NOs: 80, 82, and 102, respectively, or SEQ ID NOs: 81, 82, and 103, respectively); and (b) a VL that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 86, 77, and 87, respectively.

[0291] In certain embodiments, the first antigen-binding site that binds NKG2D comprises a VH that comprises an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 104, and a VL that comprises an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO:85. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 80 or 81, 82, and 105 or 106, respectively (e.g., SEQ ID NOs: 80, 82, and 105, respectively, or SEQ ID NOs: 81, 82, and 106, respectively). In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequencesof SEQ ID NOs: 86, 77, and 87, respectively. In certain embodiments, the first antigen-binding site comprises (a) a VH that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 80 or 81, 82, and 105 or 106, respectively (e.g., SEQ ID NOs: 80, 82, and 105, respectively, or SEQ ID NOs: 81, 82, and 106, respectively); and (b) a VL that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 86, 77, and 87, respectively.

[0292] In certain embodiments, the first antigen-binding site that binds NKG2D comprises a VH that comprises an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 107, and a VL that comprises an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO:85. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 80 or 81, 82, and 108 or 109, respectively (e.g., SEQ ID NOs: 80, 82, and 108, respectively, or SEQ ID NOs: 81, 82, and 109, respectively). In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 86, 77, and 87, respectively. In certain embodiments, the first antigen-binding site comprises (a) a VH that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 80 or 81, 82, and 108 or 109, respectively (e.g., SEQ ID NOs: 80, 82, and 108, respectively, or SEQ ID NOs: 81, 82, and 109, respectively); and (b) a VL that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 86, 77, and 87, respectively.

[0293] In certain embodiments, the first antigen-binding site that binds NKG2D comprises a VH that comprises an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 110, and a VL that comprises an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO:85. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 80 or 81, 82, and 111 or 112, respectively (e.g., SEQ ID NOs: 80, 82, and 111, respectively, or SEQ ID NOs: 81, 82, and 112, respectively). In certainembodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 86, 77, and 87, respectively. In certain embodiments, the first antigen-binding site comprises (a) a VH that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 80 or 81, 82, and 111 or 112, respectively (e.g., SEQ ID NOs: 80, 82, and 111, respectively, or SEQ ID NOs: 81, 82, and 112, respectively); and (b) a VL that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 86, 77, and 87, respectively.

[0294] In certain embodiments, the first antigen-binding site that binds NKG2D comprises a VH that comprises an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 113, and a VL that comprises an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 114.

[0295] In certain embodiments, the first antigen-binding site that binds NKG2D comprises a VH that comprises an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 116, and a VL that comprises an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 117.

[0296] The multispecific binding proteins can bind to NKG2D-expressing cells, which include but are not limited to NK cells, y6 T cells and CD8+aP T cells. Upon NKG2D binding, the multispecific binding proteins may block natural ligands, such as ULBP6 and MICA, from binding to NKG2D and activating NK cells.

[0297] The multispecific binding proteins binds to cells expressing CD 16, an Fc receptor on the surface of leukocytes including natural killer cells, macrophages, neutrophils, eosinophils, mast cells, and follicular dendritic cells. A protein of the present disclosure binds to NKG2D with an affinity of KD of 2 nM to 120 nM, e.g., 2 nM to 110 nM, 2 nM to 100 nM, 2 nM to 90 nM, 2 nM to 80 nM, 2 nM to 70 nM, 2 nM to 60 nM, 2 nM to 50 nM, 2 nM to 40 nM, 2 nM to30 nM, 2 nM to 20 nM, 2 nM to 10 nM, about 15 nM, about 14 nM, about 13 nM, about 12 nM, about 11 nM, about 10 nM, about 9 nM, about 8 nM, about 7 nM, about 6 nM, about 5 nM, about 4.5 nM, about 4 nM, about 3.5 nM, about 3 nM, about 2.5 nM, about 2 nM, about 1.5 nM, about 1 nM, between about 0.5 nM to about 1 nM, about 1 nM to about 2 nM, about 2 nM to 3 nM, about 3 nM to 4 nM, about 4 nM to about 5 nM, about 5 nM to about 6 nM, about 6 nM to about 7 nM, about 7 nM to about 8 nM, about 8 nM to about 9 nM, about 9 nM to about 10 nM, about 1 nM to about 10 nM, about 2 nM to about 10 nM, about 3 nM to about 10 nM, about 4 nM to about 10 nM, about 5 nM to about 10 nM, about 6 nM to about 10 nM, about 7 nM to about 10 nM, or about 8 nM to about 10 nM. In some embodiments, NKG2D-binding sites bind to NKG2D with a KD of 10 to 62 nM.BAFF-R Binding Site

[0298] The BAFF-R site of the multispecific binding protein disclosed herein comprises a heavy chain variable domain and a light chain variable domain.

[0299] In one aspect, the present disclosure provides multispecific binding proteins that bind to the NKG2D receptor and CD 16 receptor on natural killer cells, and BAFF-R. Table 2 lists some exemplary sequences of heavy chain variable domains and light chain variable domains that, in combination, can bind to BAFF-R.

[0300] CDR sequences are identified under Chothia numbering unless otherwise indicated.

[0301] In certain embodiments, the second antigen-binding site that binds BAFF-R (e.g., human BAFF-R) comprises an antibody heavy chain variable domain (VH) that comprises an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the VH of an antibody disclosed in Table 2, and an antibody light chain variable domain (VL) that comprises an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the VL of the same antibody disclosed in Table 2. In certain embodiments, the second antigenbinding site comprises the heavy chain CDR1, CDR2, and CDR3 and the light chain CDR1, CDR2, and CDR3, determined under Kabat (see Kabat et al., (1991) Sequences of Proteins of Immunological Interest, NIH Publication No. 91-3242, Bethesda), Chothia (see, e.g., Chothia C & Lesk A M, (1987), J Mol Biol 196: 901-917), MacCallum (see MacCallum R M et al., (1996) J Mol Biol 262: 732-745), or any other CDR determination method known in the art, of the VH and VL sequences of an antigen-binding site disclosed in Table 2. In certain embodiments, thesecond antigen-binding site comprises the heavy chain CDR1, CDR2, and CDR3 and the light chain CDR1, CDR2, and CDR3 of an antibody disclosed in Table 2.

[0302] In certain embodiments, the second antigen-binding site that binds BAFF-R comprises a VH that comprises an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 145, and a VL that comprises an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 146. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 157 or 135, 158 or 136, and 159 or 137, respectively e.g., SEQ ID NOs: 157, 158, and 159, respectively; or SEQ ID NOs: 135, 136, and 137, respectively). In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 160, 161, and 162, respectively. In certain embodiments, the second antigen-binding site comprises (a) a VH that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 157 or 135, 158 or 136, and 159 or 137, respectively (e.g., SEQ ID NOs: 157, 158, and 159, respectively; or SEQ ID NOs: 135, 136, and 137, respectively); and (b) a VL that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 160, 161, and 162, respectively. In certain embodiments, the second antigen-binding site is present as an scFv, wherein the scFv comprises an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 207 or 138.

[0303] In certain embodiments, the second antigen-binding site that binds BAFF-R comprises a VH that comprises an amino acid sequence at least 90% (e.g, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 147, and a VL that comprises an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 148. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 163, 164, and 165, respectively. In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acidsequences of SEQ ID NOs: 166, 167, and 168, respectively. In certain embodiments, the second antigen-binding site comprises (a) a VH that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 163, 164, and 165, respectively; and (b) a VL that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 166, 167, and 168, respectively. In certain embodiments, the second antigen-binding site is present as an scFv, wherein the scFv comprises an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 139 or 140.

[0304] In certain embodiments, the second antigen-binding site that binds BAFF-R comprises a VH that comprises an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 147, and a VL that comprises an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 150. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 163, 164, and 165, respectively. In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 169, 170, and 168, respectively. In certain embodiments, the second antigen-binding site comprises (a) a VH that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 163, 164, and 165, respectively; and (b) a VL that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 169, 170, and 168, respectively. In certain embodiments, the second antigen-binding site is present as an scFv, wherein the scFv comprises an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 141 or 142.

[0305] In certain embodiments, the second antigen-binding site that binds BAFF-R comprises a VH that comprises an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 151, and a VL that comprises an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identicalto SEQ ID NO: 152. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 171, 172, and 173, respectively. In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 174, 175, and 176, respectively. In certain embodiments, the second antigen-binding site comprises (a) a VH that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 171, 172, and 173, respectively; and (b) a VL that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 174, 175, and 176, respectively. In certain embodiments, the second antigen-binding site is present as an scFv, wherein the scFv comprises an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 143 or 144.

[0306] In certain embodiments, the second antigen-binding site that binds BAFF-R comprises a VH that comprises an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 153, and a VL that comprises an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 154. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 177 or 178, 179 or 180, and 181 or 182, respectively e.g., SEQ ID NOs: 177, 179, and 181, respectively; or SEQ ID NOs: 178, 180, and 182, respectively). In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 183 or 184, 185 or 186, and 187, respectively (e.g, SEQ ID NOs: 183, 185, and 187, respectively; or SEQ ID NOs: 184, 186, and 187, respectively). In certain embodiments, the second antigen-binding site comprises (a) a VH that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 177 or 178, 179 or 180, and 181 or 182, respectively (e.g, SEQ ID NOs: 177, 179, and 181, respectively; or SEQ ID NOs: 178, 180, and 182, respectively); and (b) a VL that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 183 or 184, 185 or 186, and 187, respectively (e.g., SEQ ID NOs: 183, 185, and 187, respectively; or SEQ ID NOs: 184, 186, and 187, respectively). In certain embodiments, the second antigen-binding site is present as an scFv, wherein the scFv comprises an amino acid sequence at least 90% (e.g.,at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 149 or 190.

[0307] In certain embodiments, the second antigen-binding site that binds BAFF-R comprises a VH that comprises an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 155, and a VL that comprises an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 156. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 177 or 178, 179 or 180, and 181 or 182, respectively e.g., SEQ ID NOs: 177, 179, and 181, respectively; or SEQ ID NOs: 178, 180, and 182, respectively). In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 183 or 188, 185 or 186, and 187, respectively (e.g., SEQ ID NOs: 183, 185, and 187, respectively; or SEQ ID NOs: 188, 186, and 187, respectively). In certain embodiments, the second antigen-binding site comprises (a) a VH that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 177 or 178, 179 or 180, and 181 or 182, respectively (e.g, SEQ ID NOs: 177, 179, and 181, respectively; or SEQ ID NOs: 178, 180, and 182, respectively); and (b) a VL that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 183 or 188, 185 or 186, and 187, respectively (e.g., SEQ ID NOs: 183, 185, and 187, respectively; or SEQ ID NOs: 188, 186, and 187, respectively). In certain embodiments, the second antigen-binding site is present as an scFv, wherein the scFv comprises an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 191 or 192.

[0308] In certain embodiments, the VH of the second antigen-binding site that binds BAFF-R comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 260, 249, and 261, respectively. In certain embodiments, the VL of the second antigenbinding site that binds BAFF-R comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 217, 77, and 259, respectively. In certain embodiments, the second antigen-binding site comprises (a) a VH that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 260, 249 and 261, respectively; and (b) a VL thatcomprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 217, 77, and 259, respectively.

[0309] In certain embodiments, the second antigen-binding site that binds BAFF-R comprises a VH that comprises an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 310, and a VL that comprises an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO:276. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 214, 215, and 216, respectively. In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 217, 77, and 218, respectively. In certain embodiments, the second antigen-binding site comprises (a) a VH that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 214, 215, and 216, respectively; and (b) a VL that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 217, 77, and 218, respectively.

[0310] In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 59, 60, and 218, respectively. In certain embodiments, the second antigen-binding site that binds BAFF-R comprises (a) a VH that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 214, 215, and 219, respectively; and (b) a VL that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 59, 60, and 218, respectively.

[0311] In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 220, 215, and 221, respectively. In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 217, 77, and 222, respectively. In certain embodiments, the second antigenbinding site that binds BAFF-R comprises (a) a VH that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 220, 215, and 221, respectively; and (b) a VL that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 217, 77, and 222, respectively.

[0312] In certain embodiments, the VH of the second antigen-binding site that binds BAFF-R comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 214, 215, and 226, respectively. In certain embodiments, the VL of the second antigenbinding site that binds BAFF-R comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs:217, 77, and 218, respectively. In certain embodiments, the second antigen-binding site comprises (a) a VH that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 214, 215, and 226, respectively; and (b) a VL that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 217, 77, and 218, respectively.

[0313] In certain embodiments, the second antigen-binding site that binds BAFF-R comprises a VH that comprises an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:277, and a VL that comprises an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO:276. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 214, 215, and 223, respectively. In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs:217, 77, and 218, respectively. In certain embodiments, the second antigen-binding site comprises (a) a VH that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 214, 215, and 223, respectively; and (b) a VL that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs:217, 77, and 218, respectively.

[0314] In certain embodiments, the second antigen-binding site that binds BAFF-R comprises a VH that comprises an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:278, and a VL that comprises an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO:276. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 214, 215, and 224, respectively. Incertain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs:217, 77, and 218, respectively. In certain embodiments, the second antigen-binding site comprises (a) a VH that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 214, 215, and 224, respectively; and (b) a VL that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 217, 77, and 218, respectively.

[0315] In certain embodiments, the second antigen-binding site that binds BAFF-R comprises a VH that comprises an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:279, and a VL that comprises an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO:276. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 214, 215, and 225, respectively. In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 217, 77, and 218, respectively. In certain embodiments, the second antigen-binding site comprises (a) a VH that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 214, 215, and 225, respectively; and (b) a VL that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 217, 77, and 218, respectively.

[0316] In certain embodiments, the VH of the second antigen-binding site that binds BAFF-R comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 231, 215, and 232, respectively. In certain embodiments, the VL of the second antigenbinding site that binds BAFF-R comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 217, 77, and 218, respectively. In certain embodiments, the second antigen-binding site comprises (a) a VH that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 231, 215, and 232, respectively; and (b) a VL that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 217, 77, and 218, respectively.

[0317] In certain embodiments, the second antigen-binding site that binds BAFF-R comprises a VH that comprises an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:280, and a VL that comprises an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO:276. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 227, 215, and 224, respectively. In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 217, 77, and 218, respectively. In certain embodiments, the second antigen-binding site comprises (a) a VH that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 227, 215, and 224, respectively; and (b) a VL that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 217, 77, and 218, respectively.

[0318] In certain embodiments, the second antigen-binding site that binds BAFF-R comprises a VH that comprises an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:281, and a VL that comprises an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO:276. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 228, 215, and 229, respectively. In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 217, 77, and 218, respectively. In certain embodiments, the second antigen-binding site comprises (a) a VH that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 228, 215, and 229, respectively; and (b) a VL that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 217, 77, and 218, respectively.

[0319] In certain embodiments, the second antigen-binding site that binds BAFF-R comprises a VH that comprises an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least99%, or 100%) identical to the amino acid sequence of SEQ ID NO:282, and a VL that comprises an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO:276. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 230, 215, and 224, respectively. In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 217, 77, and 218, respectively. In certain embodiments, the second antigen-binding site comprises (a) a VH that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 230, 215, and 224, respectively; and (b) a VL that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 217, 77, and 218, respectively.

[0320] In certain embodiments, the VH of the second antigen-binding site that binds BAFF-R comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 230, 233, and 236, respectively. In certain embodiments, the VL of the second antigenbinding site that binds BAFF-R comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 217, 77, and 218, respectively. In certain embodiments, the second antigen-binding site comprises (a) a VH that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 230, 233, and 236, respectively; and (b) a VL that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 217, 77, and 218, respectively.

[0321] In certain embodiments, the second antigen-binding site that binds BAFF-R comprises a VH that comprises an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:283, and a VL that comprises an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO:276. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 230, 233, and 242, respectively. In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 217, 77, and 218, respectively. In certain embodiments, the second antigen-binding site comprises (a) a VH that comprises CDR1, CDR2, and CDR3 comprising theamino acid sequences of SEQ ID NOs: 230, 233, and 242, respectively; and (b) a VL that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 217, 77, and 218, respectively.

[0322] In certain embodiments, the second antigen-binding site that binds BAFF-R comprises a VH that comprises an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:284, and a VL that comprises an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO:276. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 230, 233, and 234, respectively. In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 217, 77, and 218, respectively. In certain embodiments, the second antigen-binding site comprises (a) a VH that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 230, 233, and 234, respectively; and (b) a VL that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 217, 77, and 218, respectively.

[0323] In certain embodiments, the second antigen-binding site that binds BAFF-R comprises a VH that comprises an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:285, and a VL that comprises an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO:276. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 230, 233, and 235, respectively. In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 217, 77, and 218, respectively. In certain embodiments, the second antigen-binding site comprises (a) a VH that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 230, 233, and 235, respectively; and (b) a VL that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 217, 77, and 218, respectively.

[0324] In certain embodiments, the VH of the second antigen-binding site that binds BAFF-R comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 245, 246, and 247, respectively. In certain embodiments, the VL of the second antigenbinding site that binds BAFF-R comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 217, 77, and 259, respectively. In certain embodiments, the second antigen-binding site comprises (a) a VH that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 245, 246, and 247, respectively; and (b) a VL that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 217, 77, and 259, respectively.

[0325] In certain embodiments, the second antigen-binding site that binds BAFF-R comprises a VH that comprises an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:286, and a VL that comprises an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO:253. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 214, 233, and 237, respectively. In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 217, 77, and 249, respectively. In certain embodiments, the second antigen-binding site comprises (a) a VH that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 214, 233, and 237, respectively; and (b) a VL that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 217, 77, and 249, respectively.

[0326] In certain embodiments, the second antigen-binding site that binds BAFF-R comprises a VH that comprises an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:287, and a VL that comprises an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO:253. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 238, 239, and 240, respectively. Incertain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 217, 77, and 249, respectively. In certain embodiments, the second antigen-binding site comprises (a) a VH that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 238, 239, and 240, respectively; and (b) a VL that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 217, 77, and 249, respectively.

[0327] In certain embodiments, the second antigen-binding site that binds BAFF-R comprises a VH that comprises an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:288, and a VL that comprises an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO:253. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 241, 233, and 242, respectively. In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 217, 77, and 249, respectively. In certain embodiments, the second antigen-binding site comprises (a) a VH that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 241, 233, and 242, respectively; and (b) a VL that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 217, 77, and 249, respectively.

[0328] In certain embodiments, the second antigen-binding site that binds BAFF-R comprises a VH that comprises an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:289, and a VL that comprises an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO:289. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 243, 215, and 244, respectively. In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 217, 77, and 249, respectively. In certain embodiments, the second antigen-binding site comprises (a) a VH that comprises CDR1, CDR2, and CDR3 comprising theamino acid sequences of SEQ ID NOs: 243, 215, and 244, respectively; and (b) a VL that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 217, 77, and 249, respectively.

[0329] In certain embodiments, the VH of the second antigen-binding site that binds BAFF-R comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 256, 257 and 258, respectively. In certain embodiments, the VL of the second antigenbinding site that binds BAFF-R comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 217, 77, and 259, respectively. In certain embodiments, the second antigen-binding site comprises (a) a VH that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 256, 257 and 258, respectively; and (b) a VL that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 217, 77, and 259, respectively.

[0330] In certain embodiments, the second antigen-binding site that binds BAFF-R comprises a VH that comprises an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 250 or 252, and a VL that comprises an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO:251 or 253. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 214, 233, and 248, respectively. In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 217, 77, and 249, respectively. In certain embodiments, the second antigen-binding site comprises (a) a VH that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 214, 233, and 248, respectively; and (b) a VL that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 217, 77, and 249, respectively. In certain embodiments, the second antigen-binding site is present as an scFv, wherein the scFv comprises an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO:254 or 255.

[0331] In certain embodiments, the second antigen-binding site that binds BAFF-R comprises a VH that comprises an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:263, and a VL that comprises an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO:264. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 291, 292, and 293, respectively. In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 294, 295, and 296, respectively. In certain embodiments, the second antigen-binding site comprises (a) a VH that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 291, 292, and 293, respectively; and (b) a VL that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 294, 295, and 296, respectively.

[0332] In certain embodiments, the second antigen-binding site that binds BAFF-R comprises a VH that comprises an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:265, and a VL that comprises an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO:266. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 291, 297, and 298, respectively. In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 294, 295, and 296, respectively. In certain embodiments, the second antigen-binding site comprises (a) a VH that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 291, 297, and 298, respectively; and (b) a VL that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 294, 295, and 296, respectively.

[0333] In certain embodiments, the second antigen-binding site that binds BAFF-R comprises a VH that comprises an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least99%, or 100%) identical to the amino acid sequence of SEQ ID NO:267, and a VL that comprises an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO:268. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 299, 300, and 301, respectively. In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 302, 303, and 304, respectively. In certain embodiments, the second antigen-binding site comprises (a) a VH that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 299, 300, and 301, respectively; and (b) a VL that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 302, 303, and 304, respectively.

[0334] In certain embodiments, the second antigen-binding site that binds BAFF-R comprises a VH that comprises an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:269, and a VL that comprises an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO:262. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 305, 306, and 307, respectively. In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 308, 303, and 309, respectively. In certain embodiments, the second antigen-binding site comprises (a) a VH that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 305, 306, and 307, respectively; and (b) a VL that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 308, 303, and 309, respectively.

[0335] In certain embodiments, the second antigen-binding site that binds BAFF-R is an scFv. For example, in certain embodiments, the second antigen-binding site comprises the amino acid sequence of SEQ ID NO: 207, 138, 139, 140, 141, 142, 143, 144, 149, 190, 191, 192, 254 or 255.

[0336] Alternatively, novel antigen-binding sites that can bind to BAFF-R can be identified by screening for binding to the amino acid sequence defined by binding to the amino acid sequence defined by SEQ ID NO: 189, a variant thereof, a mature extracellular fragment thereof or a fragment containing a domain of BAFF-R.

[0337] SEQ ID NO: 189MRRGPRSLRGRDAPAPTPCVPAECFDLLVRHCVACGLLRTPRPKPAGASSPAPRTALQP QESVGAGAGEAALPLPGLLFGAPALLGLALVLALVLVGLVSWRRRQRRLRGASSAEAP DGDKDAPEPLDKVIILSPGISDATAPAWPPPGEDPGTTPPGHSVPVPATELGSTELVTTKT AGPEQQ

[0338] It is contemplated that in an scFv, a VH and a VL can be connected by a linker, e.g., (GlyGlyGlyGlySer)4 i.e. (G4S)4 linker (SEQ ID NO: 119). A skilled person in the art would appreciate that any of the other disclosed linkers (see, e.g., Table 10) may be used in an scFv having a VH and VL sequence disclosed herein (e.g., in Table 2).

[0339] In each of the foregoing embodiments, it is contemplated herein that the scFv, VH and / or VL sequences that bind BAFF-R may contain amino acid alterations (e.g., at least 1, 2, 3, 4, 5, or 10 amino acid substitutions, deletions, or additions) in the framework regions of the VH and / or VL without affecting their ability to BAFF-R. For example, it is contemplated herein that scFv, VH and / or VL sequences that bind BAFF-R may contain cysteine heterodimerization mutations, facilitating formation of a disulfide bridge between the VH and VL of the scFv.

[0340] In certain embodiments, the second antigen-binding site competes for binding to BAFF-R with a corresponding antigen-binding site described above.

[0341] In certain embodiments, the second antigen-binding site blocks interaction of BAFF-R with BAFF ligand.Fc domain

[0342] Within the Fc domain, CD16 binding is mediated by the hinge region and the CH2 domain. For example, within human IgGl, the interaction with CD16 is primarily focused on amino acid residues Asp 265 - Glu 269, Asn 297 - Thr 299, Ala 327 - He 332, Leu 234 - Ser 239, and carbohydrate residue N-acetyl-D-glucosamine in the CH2 domain (see, Sondermann et al., Nature, 406 (6793):267-273). Based on the known domains, mutations can be selected toenhance or reduce the binding affinity to CD 16, such as by using phage-displayed libraries or yeast surface-displayed cDNA libraries, or can be designed based on the known three- dimensional structure of the interaction. Accordingly, in certain embodiment, the antibody Fc domain or the portion thereof comprises a hinge and a CH2 domain.

[0343] The assembly of heterodimeric antibody heavy chains can be accomplished by expressing two different antibody heavy chain sequences in the same cell, which may lead to the assembly of homodimers of each antibody heavy chain as well as assembly of heterodimers. Promoting the preferential assembly of heterodimers can be accomplished by incorporating different mutations in the CH3 domain of each antibody heavy chain constant region as shown in US13 / 494870, US16 / 028850, US11 / 533709, US12 / 875015, US13 / 289934, US14 / 773418, US12 / 811207, US13 / 866756, US14 / 647480, US13 / 642253, and US14 / 830336. For example, mutations can be made in the CH3 domain based on human IgGl and incorporating distinct pairs of amino acid substitutions within a first polypeptide and a second polypeptide that allow these two chains to selectively heterodimerize with each other. The positions of amino acid substitutions illustrated below are all numbered according to the EU index as in Kabat (Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th Ed., United States Public Health Service, National Institutes of Health, Bethesda, entirely incorporated by reference). Those skilled in the art of antibodies will appreciate that these conventions consist of nonsequential numbering in specific regions of an immunoglobulin sequence, enabling a normalized reference to conserved positions in immunoglobulin families. Accordingly, the positions of any given immunoglobulin as defined by the EU index or by the Kabat numbering scheme will not necessarily correspond to its sequential sequence.

[0344] With knowledge of the residue number according to Kabat or EU index numbering, one of ordinary skill can apply the teachings of the art to identify amino acid sequence modifications within the present invention, according to any commonly used numbering convention. It is understood that the SEQ ID NOs provide sequential numbering of amino acids within a given polypeptide and, thus, may not conform to the corresponding amino acid numbers as provided by Kabat or EU index.

[0345] In one scenario, an amino acid substitution in the first polypeptide replaces the original amino acid with a larger amino acid, selected from arginine (R), phenylalanine (F),tyrosine (Y) or tryptophan (W), and at least one amino acid substitution in the second polypeptide replaces the original amino acid(s) with a smaller amino acid(s), chosen from alanine (A), serine (S), threonine (T), or valine (V), such that the larger amino acid substitution (a protuberance) fits into the surface of the smaller amino acid substitutions (a cavity). For example, one polypeptide can incorporate a T366W substitution, and the other can incorporate three substitutions including T366S, L368A, and Y407V.

[0346] An antibody heavy chain variable domain described in the application can optionally be coupled to an amino acid sequence at least 90% identical to an antibody constant region, such as an IgG constant region including hinge, CH2 and CH3 domains with or without CHI domain. In some embodiments, the amino acid sequence of the constant region is at least 90% identical to a human antibody constant region, such as a human IgGl constant region, an IgG2 constant region, IgG3 constant region, or IgG4 constant region. In one embodiment, the antibody Fc domain or a portion thereof sufficient to bind CD 16 comprises an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to wild-type human IgGl Fc sequenceDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 118). In some other embodiments, the amino acid sequence of the constant region is at least 90% identical to an antibody constant region from another mammal, such as rabbit, dog, cat, mouse, or horse.

[0347] In some embodiments, the antibody constant domain linked to the scFv or the Fab fragment is able to bind to CD 16. In some embodiments, the protein incorporates a portion of an antibody Fc domain (for example, a portion of an antibody Fc domain sufficient to bind CD16), wherein the antibody Fc domain comprises a hinge and a CH2 domain (for example, a hinge and a CH2 domain of a human IgGl antibody), and / or amino acid sequences at least 90% identical to amino acid sequence 234-332 of a human IgG antibody.

[0348] One or more mutations can be incorporated into the constant region as compared to human IgGl constant region, for example at Q347, Y349, L351, S354, E356, E357, K360, Q362, S364, T366, L368, K370, N390, K392, T394, D399, S400, D401, F405, Y407, K409, T411 and / or K439. Exemplary substitutions include, for example, Q347E, Q347R, Y349S, Y349K, Y349T, Y349D, Y349E, Y349C, T350V, L351K, L351D, L351Y, S354C, E356K, E357Q, E357L, E357W, K360E, K360W, Q362E, S364K, S364E, S364H, S364D, T366V, T366I, T366L, T366M, T366K, T366W, T366S, L368E, L368A, L368D, K370S, N390D, N390E, K392L, K392M, K392V, K392F, K392D, K392E, T394F, T394W, D399R, D399K, D399V, S400K, S400R, D401K, F405A, F405T, F405L, Y407A, Y407I , Y407V, K409F, K409W, K409D, K409R, T41 ID, T41 IE, K439D, and K439E.

[0349] In certain embodiments, mutations that can be incorporated into the CHI of a human IgGl constant region may be at amino acid V125, F126, P127, T135, T139, A140, F170, P171, and / or VI 73. In certain embodiments, mutations that can be incorporated into the CK of a human IgGl constant region may be at amino acid E123, Fl 16, S176, V163, S174, and / or T164.

[0350] Alternatively, amino acid substitutions could be selected from the following sets of substitutions shown in Table 3.

[0351] Alternatively, amino acid substitutions could be selected from the following sets of substitutions shown in Table 4.

[0352] Alternatively, amino acid substitutions could be selected from the following sets of substitutions shown in Table 5.

[0353] Alternatively, at least one amino acid substitution in each polypeptide chain could be selected from Table 6.

[0354] Alternatively, at least one amino acid substitution could be selected from the following sets of substitutions in Table 7, where the position(s) indicated in the First Polypeptide column is replaced by any known negatively-charged amino acid, and the position(s) indicated in the Second Polypeptide Column is replaced by any known positively-charged amino acid.

[0355] Alternatively, at least one amino acid substitution could be selected from the following set in Table 8, where the position(s) indicated in the First Polypeptide column is replaced by any known positively-charged amino acid, and the position(s) indicated in the Second Polypeptide Column is replaced by any known negatively-charged amino acid.

[0356] Alternatively, amino acid substitutions could be selected from the following sets inTable 9

[0357] Alternatively, or in addition, the structural stability of a hetero-multimeric protein may be increased by introducing S354C on either of the first or second polypeptide chain, and Y349C on the opposing polypeptide chain, which forms an artificial disulfide bridge within the interface of the two polypeptides.

[0358] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgGl (e.g., human IgGl) constant region at position T366, and wherein the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgGl (e.g., human IgGl) constant region at one or more positions selected from the group consisting of T366, L368 and Y407.

[0359] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgGl (e.g., human IgGl) constant region at one or more positions selected from the group consisting of T366, L368 and Y407, and wherein the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgGl (e.g., human IgGl) constant region at position T366.

[0360] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgGl (e.g., human IgGl) constant region at one or more positions selected from the group consisting of E357, K360, Q362, S364, L368, K370, T394, D401, F405, and T411 and wherein the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgGl (e.g., human IgGl) constant region at one or more positions selected from the group consisting of Y349, E357, S364, L368, K370, T394, D401, F405 and T411.

[0361] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgGl (e.g., human IgGl) constant region at one or more positions selected from the group consisting of Y349, E357, S364, L368, K370, T394, D401, F405 and T411 and wherein the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgGl (e.g., human IgGl) constant region at one or more positions selected from the group consisting of E357, K360, Q362, S364, L368, K370, T394, D401, F405, and T411.

[0362] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgGl (e.g., human IgGl) constant region at one or more positions selected from the group consisting of L351, D399, S400 and Y407 and wherein the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgGl (e.g., human IgGl) constant region at one or more positions selected from the group consisting of T366, N390, K392, K409 and T411.

[0363] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgGl (e.g., human IgGl) constant region at one or more positions selected from the group consisting of T366, N390, K392, K409 and T411 and wherein the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgGl (e.g., human IgGl) constant region at one or more positions selected from the group consisting of L351, D399, S400 and Y407.

[0364] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgGl (e.g., human IgGl) constant region at one or more positions selected from the group consisting of Q347, Y349, K360, and K409, and wherein the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgGl (e.g., human IgGl) constant region at one or more positions selected from the group consisting of Q347, E357, D399 and F405.

[0365] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgGl (e.g., human IgGl) constant region at one or more positions selected from the group consisting of Q347, E357, D399 and F405, and wherein the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgGl (e.g., human IgGl) constant region at one or more positions selected from the group consisting of Y349, K360, Q347 and K409.

[0366] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgGl (e.g., human IgGl)constant region at one or more positions selected from the group consisting of K370, K392, K409 and K439, and wherein the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgGl (e.g., human IgGl) constant region at one or more positions selected from the group consisting of D356, E357 and D399.

[0367] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgGl (e.g., human IgGl) constant region at one or more positions selected from the group consisting of D356, E357 and D399, and wherein the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgGl (e.g., human IgGl) constant region at one or more positions selected from the group consisting of K370, K392, K409 and K439.

[0368] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgGl (e.g., human IgGl) constant region at one or more positions selected from the group consisting of L351, E356, T366 and D399, and wherein the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgGl (e.g., human IgGl) constant region at one or more positions selected from the group consisting of Y349, L351, L368, K392 and K409.

[0369] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgGl (e.g., human IgGl) constant region at one or more positions selected from the group consisting of Y349, L351, L368, K392 and K409, and wherein the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgGl (e.g., human IgGl) constant region at one or more positions selected from the group consisting of L351, E356, T366 and D399.

[0370] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgGl (e.g., human IgGl) constant region by an S354C substitution and wherein the amino acid sequence of the otherpolypeptide chain of the antibody constant region differs from the amino acid sequence of an IgGl (e.g., human IgGl) constant region by a Y349C substitution.

[0371] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgGl (e.g., human IgGl) constant region by a Y349C substitution and wherein the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgGl (e.g., human IgGl) constant region by an S354C substitution.

[0372] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgGl (e.g., human IgGl) constant region by K360E and K409W substitutions and wherein the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgGl (e.g., human IgGl) constant region by Q347R, D399V and F405T substitutions.

[0373] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgGl (e.g., human IgGl) constant region by Q347R, D399V and F405T substitutions and wherein the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgGl (e.g., human IgGl) constant region by K360E and K409W substitutions.

[0374] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgGl (e.g., human IgGl) constant region by a T366W substitutions and wherein the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgGl (e.g., human IgGl) constant region by T366S, T368A, and Y407V substitutions.

[0375] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgGl (e.g., human IgGl) constant region by T366S, T368A, and Y407V substitutions and wherein the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgGl (e.g., human IgGl) constant region by a T366W substitution.

[0376] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgGl (e.g., human IgGl) constant region by T350V, L351Y, F405A, and Y407V substitutions and wherein the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgGl (e.g., human IgGl) constant region by T350V, T366L, K392L, and T394W substitutions.

[0377] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgGl (e.g., human IgGl) constant region by T350V, T366L, K392L, and T394W substitutions and wherein the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgGl (e.g., human IgGl) constant region by T350V, L351Y, F405A, and Y407V substitutions.

[0378] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgGl (e.g., human IgGl) constant region by an F405L substitution and wherein the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgGl (e.g., human IgGl) constant region by a K409R substitution.Exemplary multispecific binding proteins

[0379] Listed below are examples of TriNKETs comprising an antigen-binding site that binds BAFF-R and an antigen-binding site that binds NKG2D each linked to an antibody constant region, wherein the antibody constant regions include mutations that enable heterodimerization of two Fc chains.

[0380] Exemplary BAFF-R-targeting TriNKETs are contemplated in the F3’, F4, and 2- Fab formats. As described above, in the F3’ format, the antigen-binding site that binds BAFF-R is an scFv and the antigen-binding site that binds NKG2D is a Fab. In the F4 format, the antigen binding-sites that bind BAFF-R are Fab fragments and the antigen-binding site that binds NKG2D is an scFv. In each TriNKET, the scFv may comprise substitution of Cys in the VH and VL regions, facilitating formation of a disulfide bridge between the VH and VL of the scFv. In the 2-Fab format, both the antigen-binding site that binds BAFF-R and the antigen-binding site that binds NKG2D are Fabs.

[0381] The VH and VL of an scFv can be connected via a linker, e.g., a peptide linker. In certain embodiments, the peptide linker is a flexible linker. Regarding the amino acid composition of the linker, peptides are selected with properties that confer flexibility, do not interfere with the structure and function of the other domains of the proteins described in the present application, and resist cleavage from proteases. For example, glycine and serine residues generally provide protease resistance. In certain embodiments, the VL is linked N-terminal or C- terminal to the VH via a (GlyGlyGlyGlySer)4 ((G4S)4) linker (SEQ ID NO: 119).

[0382] The length of the linker (e.g., flexible linker) can be “short,” e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 amino acid residues, or “long,” e.g., at least 13 amino acid residues. In certain embodiments, the linker is 10-50, 10-40, 10-30, 10-25, 10-20, 15-50, 15-40, 15-30, 15- 25, 15-20, 20-50, 20-40, 20-30, or 20-25 amino acid residues in length.

[0383] In certain embodiments, the linker comprises or consists of a (GS)n (SEQ ID NO: 120), (GGS)n(SEQ ID NO: 121), (GGGS)n(SEQ ID NO: 122), (GGSG)n(SEQ ID NO: 123), (GGSGG)n(SEQ ID NO:124), and (GGGGS)n(SEQ ID NO:125) sequence, wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In certain embodiments, the linker comprises or consists of an amino acid sequence selected from SEQ ID NO: 119 and SEQ ID NO: 126-134, as listed in Table 10.

[0384] In the F3 ’-TriNKETs, the BAFF-R binding scFv is linked to the N-terminus of an Fc via an Ala-Ser or Gly-Ser linker. The Ala-Ser or Gly-Ser linker is included at the elbow hinge region sequence to balance between flexibility and optimal geometry. In certain embodiments, an additional amino acid sequence Thr-Lys-Gly can be added N-terminal or C-terminal to the Ala- Ser or Gly-Ser sequence at the hinge. In the F4 TriNKETs, the NKG2D-binding scFv is linked to the C-terminus of an Fc via a short linker comprising the amino acid sequence SGSGGGGS (SEQ ID NO:274).

[0385] As used herein to describe these exemplary TriNKETs, an Fc includes an antibody hinge, CH2, and CH3. In each exemplary TriNKET, the Fc domain linked to an scFv comprises the mutations of Q347R, D399V, and F405T, and the Fc domain linked to a Fab comprises matching mutations K360E and K409W for forming a heterodimer. The Fc domain linked to the scFv further includes an S354C substitution in the CH3 domain, which forms a disulfide bond with a Y349C substitution on the Fc linked to the Fab. These substitutions are bold in the sequences described in this subsection.

[0386] For example, a TriNKET described in the present disclosure is ianalumab-F3'. Ianalumab-F3' includes (a) a BAFF-R-binding scFv sequence comprising the VH and VL sequences of ianalumab described of Table 2, in the orientation of VH positioned C-terminal to VL, linked to an Fc domain and (b) an NKG2D-binding Fab fragment derived from A49MI, including a heavy chain portion comprising a heavy chain variable domain and a CHI domain, and a light chain portion comprising a light chain variable domain and a light chain constant domain, wherein the CHI domain is connected to the Fc domain. Ianalumab-F3' includes three polypeptides: scFv-ianalumab-VL-VH-Fc (SEQ ID NO: 193), A49MI-VH-CH1-Fc (SEQ ID NO: 194), and A49MLVL-CL (SEQ ID NO: 195).

[0387] scFv-ianalumab- VL- VH-Fc (SEQ ID NO: 193) (“Chain S”)DIVLTQSPATLSLSPGERATLSCRASQFILPEYLSWYQQKPGQAPRLLIYGSSSRATGVPA RFSGSGSGTDFTLTISSLEPEDFAVYYCQQFYSSPLTFGCGTKVEIKGGGGSGGGGSGGGGSGGGGSQVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWGWIRQSPGRCLEWLGR IYYRSKWYNSYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCARYQWVPKIGVF DSWGQGTLVTVSSASDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD VSHEDPEVI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLTVLHQDWLNGI<EYI<C KVSNKALPAPIEKTISKAKGQPREPRVYTLPPCRDELTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVL VSDGSFTLYSKLTVDKSRWQQGNVF SC SVMHEALHNHYTQK SLSLSPG

[0388] A49MI-VH-CH1-Fc (SEQ ID NO: 194) (“Chain H”)EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVROAPGKGLEWVSSISSSSSYIY YADSVKGRFTISRDNAKNSLYLOMNSLRAEDTAVYYCARGAPIGAAAGWFDPWGOGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPA PELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK PREEQ YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VC TLPPSRDELTENQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSW LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0389] A49MI-VL-CL (SEQ ID NO: 195) (“Chain L”)DIOMTOSPSSVSASVGDRVTITCRASOGISSWLAWYOOKPGKAPKLLIYAASSLQSGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCQQGVSFPRTFGGGTKVEIKRTVAAPSVFIFPPSD EQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0390] scFv-ianalumab-VL-VH-Fc (SEQ ID NO: 193) represents the full sequence of a BAFF-R binding scFv linked to an Fc domain via a hinge comprising Ala-Ser. The Fc domain linked to the scFv includes Q347R, D399V, and F405T substitutions for heterodimerization and an S354C substitution for forming a disulfide bond with a Y349C substitution in A49MI-VH- CHl-Fc as described below. The scFv has the amino acid sequence of SEQ ID NO:207, which includes a heavy chain variable domain of ianalumab connected to the C-terminus of a light chain variable domain of ianalumab via a (G4S)4 linker. The scFv comprises substitution of Cys in the VH and VL regions at G44 and QI 00, facilitating formation of a disulfide bridge between the VH and VL of the scFv.

[0391] A49MI-VH-CH1-Fc (SEQ ID NO: 194) represents the heavy chain portion of the Fab fragment, which comprises a heavy chain variable domain of NKG2D-binding A49MI (SEQ ID NO:95) and a CHI domain, connected to an Fc domain. The Fc domain in A49MI-VH-CH1- Fc includes a Y349C substitution in the CH3 domain, which forms a disulfide bond with an S354C substitution on the Fc in scFv-ianalumab-VL-VH-Fc. In A49MI-VH-CH1-Fc, the Fc domain also includes K360E and K409W substitutions for heterodimerization with the Fc in scFv-ianalumab-VL-VH-Fc.

[0392] A49MI-VL-CL (SEQ ID NO: 195) represents the light chain portion of the Fab fragment comprising a light chain variable domain of NKG2D-binding A49MI (SEQ ID NO:85) and a light chain constant domain.

[0393] Another TriNKET described in the present disclosure is ianalumab-2-Fab. Ianalumab-2-Fab includes (a) a BAFF-R-binding Fab fragment comprising a VH sequence and a VL sequences of ianalumab described in Table 2, including a heavy chain portion comprising a heavy chain variable domain and a CHI domain, and a light chain portion comprising a light chain variable domain and a light chain constant domain, wherein the CHI domain is connected to an Fc domain (which does not include antibody-dependent cellular cytotoxicity-enhancing mutations present in the commercial ianalumab antibody); (b) an NKG2D-binding Fab fragment derived from A49MI, including a heavy chain portion comprising a heavy chain variable domain and a CHI domain, and a light chain portion comprising a light chain variable domain and a light chain constant domain, wherein the CHI domain is connected to the Fc domain. Ianalumab-2- Fab includes four polypeptides: ianalumab-VH-CHl-Fc-Genmab, ianalumab-VL-CL, A49MI- VH-CHl-Fc, and A49MI-VL-CL-Genmab.

[0394] lanalumab-VH-CH 1-Fc-Genmab (SEQ ID NO: 196)QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWGWIRQSPGRGLEWLGRIYYRSKW YNSYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCARYQWVPKIGVFDSWGQGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPA PELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK PREEQ YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0395] lanalumab-VL-CL (SEQ ID NO : 197)DIVLTQSPATLSLSPGERATLSCRASQFILPEYLSWYQQKPGQAPRLLIYGSSSRATGVPA RFSGSGSGTDFTLTISSLEPEDFAVYYCQQFYSSPLTFGQGTKVEIKRTVAAPSVFIFPPSD EQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0396] A49MI-VH-CH1-Fc-Genmab (SEQ ID NO:213)EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVROAPGKGLEWVSSISSSSSYIY YADSVKGRFTISRDNAKNSLYLOMNSLRAEDTAVYYCARGAPIGAAAGWFDPWGOGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPA PELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK PREEQ YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV YTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS RLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0397] A49MI-VL-CL (SEQ ID NO: 195)DIOMTOSPSSVSASVGDRVTITCRASOGISSWLAWYOOKPGKAPKLLIYAASSLQSGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCQQGVSFPRTFGGGTKVEIKRTVAAPSVFIFPPSD EQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0398] lanalumab-VH-CHl-Fc-Genmab (SEQ ID NO: 196) represents the heavy chain portion of the Fab fragment, which comprises a heavy chain variable domain (SEQ ID NO: 145) of BAFF-R-binding ianalumab and a CHI domain, connected to an Fc domain. The Fc domain in ianalumab-VH-CHl-Fc-Genmab includes an F405L substitution for heterodimerization with the Fc in A49MI-VH-CH1-Fc-Genmab, which includes a K409R substitution.

[0399] lanalumab-VL-CL (SEQ ID NO: 197) represents the light chain portion of the Fab fragment comprising a light chain variable domain of BAFF-R-binding ianalumab (SEQ ID NO: 146) and a light chain constant domain.

[0400] A49MI-VH-CH1-Fc-Genmab (SEQ ID NO:213) comprises a heavy chain variable domain of NKG2D-binding A49MI (SEQ ID NO:95) and a CHI domain, connected to an Fc domain. The Fc domain in A49MI-VH-CH1-Fc-Genmab includes a K409R substitution for heterodimerization with the Fc in ianalumab-VH-CHl-Fc-Genmab, which includes an F405L substitution.

[0401] A49MI-VL-CL (SEQ ID NO: 195), as described above, comprises a light chain variable domain of NKG2D-binding A49MI (SEQ ID NO:85) and a light chain constant domain.

[0402] Another exemplary TriNKET described in the present disclosure is hCOH-l-F3’ TriNKET. hCOH-l-F3’ includes (a) a BAFF-R-binding scFv sequence derived from hCOH-1 of Table 2, in the orientation of VH positioned C-terminal to VL, linked to an Fc domain and (b) an NKG2D-binding Fab fragment derived from A49MI, including a heavy chain portion comprising a heavy chain variable domain and a CHI domain, and a light chain portion comprising a light chain variable domain and a light chain constant domain, wherein the CHI domain is connected to the Fc domain. hCOH-1 -F3' includes three polypeptides: scFv-hCOH-l-VL-VH-Fc, A49MI- VH-CHl-Fc, and A49MI-VL-CL.

[0403] scFv-hCOH-l-VL-VH-Fc (SEQ ID NO : 198) (“Chain S”)EIVLTQSPATLSLSPGERATLSCRASESVDNYGISFLNWFQQKPGQAPRLLIYAASNRATG IPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSKEVPWTFGCGTKVEIKGGGGSGGGGS GGGGSGGGGSQVQLQESGPGLVKPSQTLSLTCTVSGDSITSGYWNWIRQHPGKCLEYIG YISYSGSTYYNPSLKSRVTISRDTSKNQFSLKLSSVTAADTAVYYCASPNYPFYAMDYW GQGTLVTVSSASDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH EDPEVI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLTVLHQDWLNGI<EYI<CI<VS NKALPAPIEKTISKAKGQPREPRVYTLPPCRDELTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLVSDGSFTL YSKLTVDKSRWQQGNVF SC S VMHEALHNHYTQKSLS LSPG

[0028] scFv-hCOH-l-VL-VH-Fc (SEQ ID NO: 198) represents the full sequence of a BAFF- R binding scFv linked to an Fc domain via a hinge comprising Ala-Ser. The Fc domain linked to the scFv includes Q347R, D399V, and F405T substitutions for heterodimerization and an S354C substitution for forming a disulfide bond with a Y349C substitution in A49MI-VH-CH1-Fc as described below. The scFv has the amino acid sequence of SEQ ID NO: 149, which includes aheavy chain variable domain of hCOH-1 connected to the C-terminus of a light chain variable domain of hCOH-1 via a (G4S)4 linker. The scFv comprises substitution of Cys in the VH and VL regions at G44 and G100, facilitating formation of a disulfide bridge between the VH and VL of the scFv.

[0404] A49MI-VH-CH1-Fc (SEQ ID NO: 194), as described above, comprises a heavy chain variable domain of NKG2D-binding A49MI (SEQ ID NO:95) and a CHI domain, connected to an Fc domain. The Fc domain in A49MI-VH-CH1-Fc includes a Y349C substitution in the CH3 domain, which forms a disulfide bond with an S354C substitution on the Fc in scFv-hCOH-l-VL-VH-Fc. In A49MI-VH-CH1-Fc, the Fc domain also includes K360E and K409W substitutions for heterodimerization with the Fc in scFv-hCOH-l-VL-VH-Fc.

[0405] A49MI-VL-CL (SEQ ID NO: 195), as described above, comprises a light chain variable domain of NKG2D-binding A49MI (SEQ ID NO:85) and a light chain constant domain.

[0406] Another TriNKET described in the present disclosure is hCOH-l-2-Fab. hCOH-1-2- Fab includes (a) a BAFF-R-binding Fab fragment derived from hCOH-1, including a heavy chain portion comprising a heavy chain variable domain and a CHI domain, and a light chain portion comprising a light chain variable domain and a light chain constant domain, wherein the CHI domain is connected to an Fc domain; (b) an NKG2D-binding Fab fragment derived from A49MI, including a heavy chain portion comprising a heavy chain variable domain and a CHI domain, and a light chain portion comprising a light chain variable domain and a light chain constant domain, wherein the CHI domain is connected to the Fc domain. hCOH-l-2-Fab includes four polypeptides: hCOH-1 -VH-CHl-Fc-Genmab, hCOH-l-VL-CL, A49MI-VH-CH1- Fc-Genmab, and A49MI-VL-CL.

[0407] hCOH-1 -VH-CHl-Fc-Genmab (SEQ ID NO:208)QVQLQESGPGLVKPSQTLSLTCTVSGDSITSGYWNWIRQHPGKGLEYIGYISYSGSTYYN PSLKSRVTISRDTSKNQFSLKLSSVTAADTAVYYCASPNYPFYAMDYWGQGTLVTVSSA STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGP SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLLYSKLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0408] hCOH-l-VL-CL (SEQ ID NO:209)EIVLTQSPATLSLSPGERATLSCRASESVDNYGISFLNWFQQKPGQAPRLLIYAASNRATG IPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSKEVPWTFGGGTKVEIKRTVAAPSVFIFP PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST LTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0409] hCOH-l-VH-CHl-Fc-Genmab (SEQ ID NO:208) represents the heavy chain portion of the Fab fragment, which comprises a heavy chain variable domain of BAFF-R-binding hCOH-1 (SEQ ID NO: 153) and a CHI domain, connected to an Fc domain. The Fc domain in hCOH-l-VH-CHl-Fc-Genmab includes an F405L substitution for heterodimerization with theFc in A49MI-VH-CH1-Fc-Genmab, which includes a K409R substitution.

[0410] hCOH-l-VL-CL (SEQ ID NO:209) represents the light chain portion of the Fab fragment comprising a light chain variable domain of BAFF-R-binding hCOH-1 (SEQ ID NO: 154) and a light chain constant domain.

[0411] A49MI-VH-CH1-Fc-Genmab (SEQ ID NO:213) comprises a heavy chain variable domain of NKG2D-binding A49MI (SEQ ID NO:95) and a CHI domain, connected to an Fc domain. The Fc domain in A49MI-VH-CH1-Fc-Genmab includes a K409R substitution for heterodimerization with the Fc in hCOH-l-VH-CHl-Fc-Genmab, which includes an F405L substitution.

[0412] A49MI-VL-CL (SEQ ID NO: 195) comprises a light chain variable domain of NKG2D-binding A49MI (SEQ ID NO:85) and a light chain constant domain.

[0413] Another exemplary TriNKET described in the present disclosure is hCOH-2-F3’. hCOH-2-F3’ includes (a) a BAFF-R-binding scFv sequence derived from hCOH-2 of Table 2, in the orientation of VH positioned C-terminal to VL, linked to an Fc domain and (b) an NKG2D- binding Fab fragment derived from A49MI, including a heavy chain portion comprising a heavy chain variable domain and a CHI domain, and a light chain portion comprising a light chain variable domain and a light chain constant domain, wherein the CHI domain is connected to theFc domain. hCOH-2-F3' includes three polypeptides: scFv-hCOH-l-VL-VH-Fc, A49MI-VH- CHl-Fc, and A49MI-VL-CL.

[0414] scFv-hCOH-2-VL-VH-Fc (SEQ ID NO :210) (“Chain S”)DIVLTQSPATLSLSPGERATLSCRASESVDNYGISFMNWFQQKPGQAPRLLIYAASNRATG IPARF SGSGSGTDFTLTIS SLEPEDFAVYYCQQ SKEVPWTFGCGTKVEIKGGGGSGGGGSG GGGSGGGGSEVQLQESGPGLVKPSQTLSLTCTVSGDSITSGYWNWIRQHPGKCLEYIGYI SYSGSTYYNPSLKSRVTISRDTSKNQYSLKLSSVTAADTAVYYCASPNYPFYAMDYWGQ GTLVTVSSASDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP EVI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLTVLHQDWLNGI<EYI<CI<VSNI<A LPAPIEKTISKAKGQPREPRVYTLPPCRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQP ENNYKTTPPVLVSDGSFTLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP G

[0415] scFv-hCOH-2-VL-VH-Fc (SEQ ID NO:210) represents the full sequence of a BAFF-R binding scFv linked to an Fc domain via a hinge comprising Ala-Ser. The Fc domain linked to the scFv includes Q347R, D399V, and F405T substitutions for heterodimerization and an S354C substitution for forming a disulfide bond with a Y349C substitution in A49MI-VH- CHl-Fc as described below. The scFv has the amino acid sequence of SEQ ID NO: 191, which includes a heavy chain variable domain of hCOH-2 connected to the C-terminus of a light chain variable domain of hCOH-2 via a (G4S)4 linker. The scFv comprises substitution of Cys in the VH and VL regions at G44 and G100, facilitating formation of a disulfide bridge between the VH and VL of the scFv.

[0416] A49MI-VH-CH1-Fc (SEQ ID NO: 194) represents the heavy chain portion of theFab fragment, which comprises a heavy chain variable domain of NKG2D-binding A49MI (SEQ ID NO:95) and a CHI domain, connected to an Fc domain. The Fc domain in A49MI-VH-CH1- Fc includes a Y349C substitution in the CH3 domain, which forms a disulfide bond with an S354C substitution on the Fc in scFv-hCOH-2-VL-VH-Fc. In A49MI-VH-CH1-Fc, the Fc domain also includes K360E and K409W substitutions for heterodimerization with the Fc in scFv-hCOH-2-VL-VH-Fc.

[0417] A49MI-VL-CL (SEQ ID NO: 195) represents the light chain portion of the Fab fragment comprising a light chain variable domain of NKG2D-binding A49MI (SEQ ID NO:85) and a light chain constant domain.

[0418] Another TriNKET described in the present disclosure is hCOH-2-2-Fab. hCOH-2-2- Fab includes (a) a BAFF-R-binding Fab fragment derived from hCOH-2, including a heavy chain portion comprising a heavy chain variable domain and a CHI domain, and a light chain portion comprising a light chain variable domain and a light chain constant domain, wherein the CHI domain is connected to an Fc domain; (b) an NKG2D-binding Fab fragment derived from A49MI, including a heavy chain portion comprising a heavy chain variable domain and a CHI domain, and a light chain portion comprising a light chain variable domain and a light chain constant domain, wherein the CHI domain is connected to the Fc domain. hCOH-2-2-Fab includes four polypeptides: hCOH-2-VH-CHl-Fc-Genmab, hCOH-2-VL-CL, A49MI-VH-CH1- Fc-Genmab, and A49MI-VL-CL.

[0419] hCOH-2-VH-CHl-Fc-Genmab (SEQ ID NO: 199) EVQLQESGPGLVKPSQTLSLTCTVSGDSITSGYWNWIRQHPGKGLEYIGYISYSGSTYYN PSLKSRVTISRDTSKNQYSLKLSSVTAADTAVYYCASPNYPFYAMDYWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR DELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLLYSKLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0420] hCOH-2-VL-CL (SEQ ID NO:200) DIVLTQSPATLSLSPGERATLSCRASESVDNYGISFMNWFQQKPGQAPRLLIYAASNRAT GIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSKEVPWTFGGGTKVEIKRTVAAPSVFI FPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLS STLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0421] hCOH-2-VH-CHl-Fc-Genmab (SEQ ID NO: 199) represents the heavy chain portion of the Fab fragment, which comprises a heavy chain variable domain of BAFF-R-binding hCOH-2 (SEQ ID NO: 155) and a CHI domain, connected to an Fc domain. The Fc domain inhCOH-2-VH-CHl-Fc-Genmab includes an F405L substitution for heterodimerization with the Fc in A49MI-VH-CH1-Fc-Genmab, which includes a K409R substitution.

[0422] hCOH-2-VL-CL (SEQ ID NO:200) represents the light chain portion of the Fab fragment comprising a light chain variable domain of BAFF-R-binding hCOH-2 (SEQ ID NO: 156) and a light chain constant domain.

[0423] A49MI-VH-CH1-Fc-Genmab (SEQ ID NO:213) comprises a heavy chain variable domain of NKG2D-binding A49MI (SEQ ID NO:95) and a CHI domain, connected to an Fc domain. The Fc domain in A49MI-VH-CH1-Fc-Genmab includes a K409R substitution for heterodimerization with the Fc in hCOH-2-VH-CHl-Fc-Genmab, which includes an F405L substitution.

[0424] A49MI-VL-CL (SEQ ID NO: 195) comprises a light chain variable domain of NKG2D-binding A49MI (SEQ ID NO:85) and a light chain constant domain.

[0425] Another exemplary TriNKET described in the present disclosure is V3-46s-F3’. V3- 46s-F3’ includes (a) a BAFF-R-binding scFv sequence derived from V3-46s of Table 2, in the orientation of VH positioned C-terminal to VL, linked to an Fc domain and (b) an NKG2D- binding Fab fragment derived from A49MI, including a heavy chain portion comprising a heavy chain variable domain and a CHI domain, and a light chain portion comprising a light chain variable domain and a light chain constant domain, wherein the CHI domain is connected to the Fc domain. V3-46s-F3' includes three polypeptides: scFv-hCOH-l-VL-VH-Fc, A49MI-VH- CHl-Fc, and A49MI-VL-CL.

[0426] scFv-V3-46s-VL-VH-Fc (SEQ ID NO:201) (“Chain S”) DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSR FSGSGSGTDFTLTISSLQPEDFATYYCQQSQISPPTFGCGTKVEIKGGGGSGGGGSGGGGS GGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTISSSSIHWVRQAPGKCLEWVAWVLPS VGFTDYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRVCYNRLGVCAGGM DYWGQGTLVTVSSASDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD VSHEDPEVI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLTVLHQDWLNGI<EYI<C KVSNKALPAPIEKTISKAKGQPREPRVYTLPPCRDELTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVL VSDGSFTLYSKLTVDKSRWQQGNVF SC SVMHEALHNHYTQK SLSLSPG

[0427] scFv-V3-46s-VL-VH-Fc (SEQ ID NO:201) represents the full sequence of a BAFFIN binding scFv linked to an Fc domain via a hinge comprising Ala-Ser. The Fc domain linked to the scFv includes Q347R, D399V, and F405T substitutions for heterodimerization and an S354C substitution for forming a disulfide bond with a Y349C substitution in A49MI-VH-CH1-Fc as described below. The scFv has the amino acid sequence of SEQ ID NO: 139, which includes a heavy chain variable domain of V3-46s connected to the C-terminus of a light chain variable domain of V3-46s via a (G4S)4 linker. The scFv comprises substitution of Cys in the VH and VL regions at G44 and QI 00, facilitating formation of a disulfide bridge between the VH and VL of the scFv.

[0428] A49MI-VH-CH1-Fc (SEQ ID NO: 194) represents the heavy chain portion of the Fab fragment, which comprises a heavy chain variable domain of NKG2D-binding A49MI (SEQ ID NO:95) and a CHI domain, connected to an Fc domain. The Fc domain in A49MI-VH-CH1- Fc includes a Y349C substitution in the CH3 domain, which forms a disulfide bond with an S354C substitution on the Fc in scFv-V3-46s-VL-VH-Fc. In A49MI-VH-CH1-Fc, the Fc domain also includes K360E and K409W substitutions for heterodimerization with the Fc in scFv-V3- 46s-VL-VH-Fc.

[0429] A49MI-VL-CL (SEQ ID NO: 195) represents the light chain portion of the Fab fragment comprising a light chain variable domain of NKG2D-binding A49MI (SEQ ID NO:85) and a light chain constant domain.

[0430] Another TriNKET described in the present disclosure is V3-46s-2-Fab. V3-46s-2- Fab includes (a) a BAFF-R-binding Fab fragment derived from V3-46s, including a heavy chain portion comprising a heavy chain variable domain and a CHI domain, and a light chain portion comprising a light chain variable domain and a light chain constant domain, wherein the CHI domain is connected to an Fc domain; (b) an NKG2D-binding Fab fragment derived from A49MI, including a heavy chain portion comprising a heavy chain variable domain and a CHI domain, and a light chain portion comprising a light chain variable domain and a light chain constant domain, wherein the CHI domain is connected to the Fc domain. V3-46s-2-Fab includes four polypeptides: V3-46s-VH-CHl-Fc-Genmab, V3-46s-VL-CL, A49MI-VH-CH1-Fc- Genmab, and A49MI-VL-CL.

[0431] V3-46s-VH-CHl-Fc-Genmab (SEQ ID NO:202)EVQLVESGGGL VQPGGSLRLSC AASGFTIS S S SMWVRQAPGKGLEW VAWVLPS VGFTD YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRVCYNRLGVCAGGMDYW GQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG VHTFP AVLQS SGL YSLS S VVTVPS S SLGTQT YICNVNHKPSNTKVDKKVEPKSCDKTHTC PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP REPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG SFLLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0432] V3-46s-VL-CL (SEQ ID NO:203)DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCQQSQISPPTFGQGTKVEIKRTVAAPSVFIFPPSDE QLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLS KADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0433] V3-46s-VH-CHl-Fc-Genmab (SEQ ID NO:202) represents the heavy chain portion of the Fab fragment, which comprises a heavy chain variable domain of BAFF-R-binding V3- 46s (SEQ ID NO: 147) and a CHI domain, connected to an Fc domain. The Fc domain in V3- 46s-VH-CHl-Fc-Genmab includes an F405L substitution for heterodimerization with the Fc in A49MI-VH-CH1-Fc-Genmab, which includes a K409R substitution.

[0434] V3-46s-VL-CL (SEQ ID NO:203) represents the light chain portion of the Fab fragment comprising a light chain variable domain of BAFF-R-binding V3-46s (SEQ ID NO: 148) and a light chain constant domain.

[0435] A49MI-VH-CH1-Fc-Genmab (SEQ ID NO:213) comprises a heavy chain variable domain of NKG2D-binding A49MI (SEQ ID NO:95) and a CHI domain, connected to an Fc domain. The Fc domain in A49MI-VH-CH1-Fc-Genmab includes a K409R substitution for heterodimerization with the Fc in V3-46s-VH-CHl-Fc-Genmab, which includes an F405L substitution.

[0436] A49MI-VL-CL (SEQ ID NO: 195) comprises a light chain variable domain of NKG2D-binding A49MI (SEQ ID NO:85) and a light chain constant domain.

[0437] Another exemplary TriNKET described in the present disclosure is V3-46s-42-F3’. V3-46s-42-F3’ includes (a) a BAFF-R-binding scFv sequence derived from V3-46s-42 of Table 2, in the orientation of VH positioned C-terminal to VL, linked to an Fc domain and (b) an NKG2D-binding Fab fragment derived from A49MI, including a heavy chain portion comprising a heavy chain variable domain and a CHI domain, and a light chain portion comprising a light chain variable domain and a light chain constant domain, wherein the CHI domain is connected to the Fc domain. V3-46s-42-F3' includes three polypeptides: scFv-V3-46s-42-VL-VH-Fc, A49MI-VH-CH1-Fc, and A49MI-VL-CL.

[0438] scFv-V3-46s-42-VL-VH-Fc (SEQ ID NO :204) (“Chain S”)DIQMTQSPSSLSASVGDRVTITCRASEDISTAVAWYQQKPGKAPKLLIYAASFLYSGVPSR FSGSGSGTDFTLTISSLQPEDFATYYCQQSQISPPTFGCGTKVEIKGGGGSGGGGSGGGGS GGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTISSSSIHWVRQAPGKCLEWVAWVLPS VGFTDYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRVCYNRLGVCAGGM DYWGQGTLVTVSSASDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD VSHEDPEVI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLTVLHQDWLNGI<EYI<C KVSNKALPAPIEKTISKAKGQPREPRVYTLPPCRDELTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVL VSDGSFTLYSKLTVDKSRWQQGNVF SC SVMHEALHNHYTQK SLSLSPG

[0439] scFv-V3-46s-42-VL-VH-Fc (SEQ ID NO:204) represents the full sequence of a BAFF-R binding scFv linked to an Fc domain via a hinge comprising Ala-Ser. The Fc domain linked to the scFv includes Q347R, D399V, and F405T substitutions for heterodimerization and an S354C substitution for forming a disulfide bond with a Y349C substitution in A49MI-VH- CHl-Fc as described below. The scFv has the amino acid sequence of SEQ ID NO: 141, which includes a heavy chain variable domain of V3-46s-42 connected to the C-terminus of a light chain variable domain of V3-46s-42 via a (G4S)4 linker. The scFv comprises substitution of Cys in the VH and VL regions at G44 and QI 00, facilitating formation of a disulfide bridge between the VH and VL of the scFv.

[0440] A49MI-VH-CH1-Fc (SEQ ID NO: 194) represents the heavy chain portion of the Fab fragment, which comprises a heavy chain variable domain of NKG2D-binding A49MI (SEQ ID NO:95) and a CHI domain, connected to an Fc domain. The Fc domain in A49MI-VH-CH1-Fc includes a Y349C substitution in the CH3 domain, which forms a disulfide bond with an S354C substitution on the Fc in scFv-V3-46s-42-VL-VH-Fc. In A49MI-VH-CH1-Fc, the Fc domain also includes K360E and K409W substitutions for heterodimerization with the Fc in scFv- V3 -46s-42- VL- VH-Fc.

[0441] A49MI-VL-CL (SEQ ID NO: 195) represents the light chain portion of the Fab fragment comprising a light chain variable domain of NKG2D-binding A49MI (SEQ ID NO:85) and a light chain constant domain.

[0442] Another TriNKET described in the present disclosure is V3-46s-42-2-Fab. V3-46s- 42-2-Fab includes (a) a BAFF-R-binding Fab fragment derived from V3-46s-42, including a heavy chain portion comprising a heavy chain variable domain and a CHI domain, and a light chain portion comprising a light chain variable domain and a light chain constant domain, wherein the CHI domain is connected to an Fc domain; (b) an NKG2D-binding Fab fragment derived from A49MI, including a heavy chain portion comprising a heavy chain variable domain and a CHI domain, and a light chain portion comprising a light chain variable domain and a light chain constant domain, wherein the CHI domain is connected to the Fc domain. V3-46s-42-2- Fab includes four polypeptides: V3-46s-42-VH-CHl-Fc-Genmab, V3-46s-42-VL-CL, A49MI- VH-CHl-Fc-Genmab, and A49MI-VL-CL.

[0443] V3-46s-42-VH-CHl-Fc-Genmab (SEQ ID NO :202)EVQLVESGGGL VQPGGSLRLSC AASGFTIS S S SMWVRQAPGKGLEW VAWVLPS VGFTD YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRVCYNRLGVCAGGMDYW GQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG VHTFP AVLQS SGL YSLS S VVTVPS S SLGTQT YICNVNHKPSNTKVDKKVEPKSCDKTHTC PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP REPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG SFLLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0444] V3-46s-42-VL-CL (SEQ ID NO:206)DIQMTQSPSSLSASVGDRVTITCRASEDISTAVAWYQQKPGKAPKLLIYAASFLYSGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCQQSQISPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLS KADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0445] V3-46s-42-VH-CHl-Fc-Genmab (SEQ ID NO:205) represents the heavy chain portion of the Fab fragment, which comprises a heavy chain variable domain of BAFF-R-binding V3-46s-42 (SEQ ID NO: 147) and a CHI domain, connected to an Fc domain. The Fc domain in V3-46s-42-VH-CHl-Fc includes an F405L substitution for heterodimerization with the Fc in A49MI-VH-CH1-Fc-Genmab, which includes a K409R substitution.

[0446] V3-46s-42-VL-CL (SEQ ID NO:206) represents the light chain portion of the Fab fragment comprising a light chain variable domain of BAFF-R-binding V3-46s-42 (SEQ ID NO: 150) and a light chain constant domain.

[0447] A49MI-VH-CH1-Fc-Genmab (SEQ ID NO:213) comprises a heavy chain variable domain of NKG2D-binding A49MI (SEQ ID NO:95) and a CHI domain, connected to an Fc domain. The Fc domain in A49MI-VH-CH1-Fc-Genmab includes a K409R substitution for heterodimerization with the Fc in V3-46s-42-VH-CHl-Fc-Genmab, which includes an F405L substitution.

[0448] A49MI-VL-CL (SEQ ID NO: 195) comprises a light chain variable domain of NKG2D-binding A49MI (SEQ ID NO:85) and a light chain constant domain.

[0449] Another exemplary TriNKET described in the present disclosure is Hu9.1-73-F3’ TriNKET. Hu9.1-73-F3’ TriNKET includes (a) a BAFF-R-binding scFv sequence derived from Hu9.1-73 of Table 2, in the orientation of VH positioned C-terminal to VL, linked to an Fc domain and (b) an NKG2D-binding Fab fragment derived from A49MI, including a heavy chain portion comprising a heavy chain variable domain and a CHI domain, and a light chain portion comprising a light chain variable domain and a light chain constant domain, wherein the CHI domain is connected to the Fc domain. Hu9.1-73-F3' includes three polypeptides: scFv-Hu9.1- 73-VL-VH-Fc, A49MI-VH-CH1-Fc, and A49MI-VL-CL.

[0450] scFv-Hu9.1-73-VL-VH-Fc (SEQ ID NO:211) (“Chain S”)DIQMTQSPSSLSASVGDRVTITCKSSQSLLYSSNQNNYLAWYQQKPGKAPKLLIYWAQH LDSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYTYPYTFGCGTKVEIKGGGGSG GGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGLPMAGFYTSWVRQAPGKCLEWVGFIRDKANGYTTEYNPSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAQVR RALDYWGQGTLVTVSSASDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKE YKCKVSNKALPAPIEKTISKAKGQPREPRVYTLPPCRDELTKNQVSLTCLVKGFYPSDIA VEWESNGQPENNYKTTPPVLVSDGSFTLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPG

[0451] scFv-Hu9.1-73-VL-VH-Fc (SEQ ID NO:211) represents the full sequence of aBAFF-R binding scFv linked to an Fc domain via a hinge comprising Ala-Ser. The Fc domain linked to the scFv includes Q347R, D399V, and F405T substitutions for heterodimerization and an S354C substitution for forming a disulfide bond with a Y349C substitution in A49MI-VH- CHl-Fc as described below. The scFv has the amino acid sequence of SEQ ID NO: 143, which includes a heavy chain variable domain of scFv-Hu9.1-73 connected to the C-terminus of a light chain variable domain of scFv-Hu9.1-73 via a (G4S)4 linker. The scFv comprises substitution of Cys in the VH and VL regions at G44 and QI 00, facilitating formation of a disulfide bridge between the VH and VL of the scFv.

[0452] A49MI-VH-CH1-Fc (SEQ ID NO: 194) represents the heavy chain portion of the Fab fragment, which comprises a heavy chain variable domain of NKG2D-binding A49MI (SEQ ID NO:95) and a CHI domain, connected to an Fc domain. The Fc domain in A49MI-VH-CH1- Fc includes a Y349C substitution in the CH3 domain, which forms a disulfide bond with an S354C substitution on the Fc in scFv-Hu9.1-73-VL-VH-Fc. In A49MI-VH-CH1-Fc, the Fc domain also includes K360E and K409W substitutions for heterodimerization with the Fc in scFv-Hu9.1-73-VL-VH-Fc.

[0453] A49MI-VL-CL (SEQ ID NO: 195) represents the light chain portion of the Fab fragment comprising a light chain variable domain of NKG2D-binding A49MI (SEQ ID NO:85) and a light chain constant domain.

[0454] Another TriNKET described in the present disclosure is Hu9.1-73-2 -Fab. Hu9.1-73- 2-Fab includes (a) a BAFF-R-binding Fab fragment derived from Hu9.1-73, including a heavy chain portion comprising a heavy chain variable domain and a CHI domain, and a light chain portion comprising a light chain variable domain and a light chain constant domain, wherein the CHI domain is connected to an Fc domain; (b) an NKG2D-binding Fab fragment derived fromA49MI, including a heavy chain portion comprising a heavy chain variable domain and a CHI domain, and a light chain portion comprising a light chain variable domain and a light chain constant domain, wherein the CHI domain is connected to the Fc domain. Hu9.1-73-2-Fab includes four polypeptides: Hu9.1-73-VH-CHl-Fc-Genmab, Hu9.1-73-VL-CL, A49MI-VH- CHl-Fc-Genmab, and A49MI-VL-CL.

[0455] Hu9.1-73-VH-CHl-Fc-Genmab (SEQ ID NO:212)EVQLVESGGGLVQPGGSLRLSCAASGLPMAGFYTSWVRQAPGKGLEWVGFIRDKANG YTTEYNPSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAQVRRALDYWGQGTLV TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE LLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLLYSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0456] Hu9.1-73-VL-CL (SEQ ID NO:205)DIQMTQSPS SLS AS VGDRVTITCKS SQ SLL YS SNQNNYL AWYQQKPGKAPKLLIYWAQH LDSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYTYPYTFGQGTKVEIKRTVAAP SVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDST YSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0457] Hu9.1-73-VH-CHl-Fc-Genmab (SEQ ID NO:212) represents the heavy chain portion of the Fab fragment, which comprises a heavy chain variable domain of BAFF-R-binding Hu9.1-73 (SEQ ID NO:151) and a CHI domain, connected to an Fc domain. The Fc domain in Hu9.1-73-VH-CHl-Fc includes an F405L substitution for heterodimerization with the Fc in A49MI-VH-CH1-Fc-Genmab, which includes a K409R substitution.

[0458] Hu9.1-73-VL-CL (SEQ ID NO:205) represents the light chain portion of the Fab fragment comprising a light chain variable domain of BAFF-R-binding Hu9.1-73 (SEQ ID NO: 152) and a light chain constant domain.

[0459] A49MI-VH-CH1-Fc-Genmab (SEQ ID NO:213) comprises a heavy chain variable domain of NKG2D-binding A49MI (SEQ ID NO:95) and a CHI domain, connected to an Fc domain. The Fc domain in A49MI-VH-CH1-Fc-Genmab includes a K409R substitution forheterodimerization with the Fc in Hu9.1-73-VH-CHl-Fc-Genmab, which includes an F405L substitution.

[0460] A49MI-VL-CL (SEQ ID NO: 195) comprises a light chain variable domain ofNKG2D-binding A49MI (SEQ ID NO:85) and a light chain constant domain.

[0461] Another example of a TriNKET described in the present disclosure is AB1424 / 1612-F3'. AB1424 / 1612-F3' includes (a) a BAFF-R-binding scFv sequence derived from AB1424 / 1612 (with cysteine heterodimerization mutations for disulfide bridge formation) of Table 2, in the orientation of VH positioned N-terminal to VL, linked to an Fc domain and (b) an NKG2D-binding Fab fragment derived from A49MI, including a heavy chain portion comprising a heavy chain variable domain and a CHI domain, and a light chain portion comprising a light chain variable domain and a light chain constant domain, wherein the CHI domain is connected to the Fc domain. AB1424 / 1612-F3' includes three polypeptides: scFv- AB1424 / 1612-VL-VH-Fc (SEQ ID NO: 193), A49MI-VH-CH1-Fc (SEQ ID NO: 194), and A49MI-VL-CL (SEQ ID NO: 195).

[0462] scFv-AB1424 / 1612-VH-VL-Fc (SEQ ID NO:270) (“Chain S”)EVQLVQSGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKCLEWVAVIWYDASN KYYGDSVKGRFTISRDNSKNTLYLQMNSLRDEDTAVYYCARRFTHLRGQYIEDYGLDV WGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSEIVLTQSPSSLSASVGDRVTITCRASQSI SSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQ QSYSIPLTFGCGTKVEIKGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKE YKCKVSNKALPAPIEKTISKAKGQPREPRVYTLPPCRDELTKNQVSLTCLVKGFYPSDIA VEWESNGQPENNYKTTPPVLVSDGSFTLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPG

[0463] A49MI-VH-CH1-Fc (SEQ ID NO: 194) (“Chain H”) EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVROAPGKGLEWVSSISSSSSYIY YADSVKGRFTISRDNAKNSLYLOMNSLRAEDTAVYYCARGAPIGAAAGWFDPWGOGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPA PELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VC TLPPSRDELTENQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSW LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0464] A49MI-VL-CL (SEQ ID NO: 195) (“Chain L”) DIOMTOSPSSVSASVGDRVTITCRASOGISSWLAWYOOKPGKAPKLLIYAASSLQSGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCQQGVSFPRTFGGGTKVEIKRTVAAPSVFIFPPSD EQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0465] scFv- AB 1424 / 1612- VH-VL-Fc (SEQ ID NO:270) represents the full sequence of aBAFF-R binding scFv linked to an Fc domain via a hinge comprising Ala-Ser. The Fc domain linked to the scFv includes Q347R, D399V, and F405T substitutions for heterodimerization and an S354C substitution for forming a disulfide bond with a Y349C substitution in A49MI-VH- CHl-Fc as described below. The scFv has the amino acid sequence of SEQ ID NO:254, which includes a heavy chain variable domain of AB 1424 / 1612 connected to the C-terminus of a light chain variable domain of AB 1424 / 1612 via a (G4S)4 linker. The scFv comprises substitution of cysteine in the VH and VL regions at G44 and G100, facilitating formation of a disulfide bridge between the VH and VL of the scFv.

[0466] A49MI-VH-CH1-Fc (SEQ ID NO: 194) represents the heavy chain portion of the Fab fragment, which comprises a heavy chain variable domain of NKG2D-binding A49MI (SEQ ID NO:95) and a CHI domain, connected to an Fc domain. The Fc domain in A49MI-VH-CH1- Fc includes a Y349C substitution in the CH3 domain, which forms a disulfide bond with an S354C substitution on the Fc in scFv-AB1424 / 1612-VL-VH-Fc. In A49MI-VH-CH1-Fc, the Fc domain also includes K360E and K409W substitutions for heterodimerization with the Fc in scFv- AB1424 / 1612-VL-VH-Fc.

[0467] A49MI-VL-CL (SEQ ID NO: 195) represents the light chain portion of the Fab fragment comprising a light chain variable domain of NKG2D-binding A49MI (SEQ ID NO:85) and a light chain constant domain.

[0468] Another example of a TriNKET described in the present disclosure isAB1424 / 1612-F4. AB1424 / 1612-F4 includes (a) two BAFF-R-binding Fab fragments derived from AB1424 / 1612 of Table 2, each including a heavy chain portion comprising a heavy chainvariable domain and a CHI domain, and a light chain portion comprising a light chain variable domain and a light chain constant domain, wherein the CHI domain is connected to the Fc domain and (b) an NKG2D-binding scFv sequence derived from A49MI linked to the C- terminus of the Fc domain, in the orientation of VH positioned C-terminal to VL. AB1424 / 1612- F4 includes four polypeptides: a first polypeptide comprising AB 1424 / 1612-VH-CH1-CH2- CH3-A49MI-scFv (SEQ ID NO:271), a second polypeptide comprising AB- 1424 / 1612-VH- CH1-CH2-CH3 (SEQ ID NO:272), and a third and fourth polypeptide each comprising AB1424 / 1612-VL-CL (SEQ ID NO:273).

[0469] AB1424 / 1612-VH-CHl-CH2-CH3-A49MI-scFv Q ID NO:271) (Chain “M”)EVQLVQSGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWYDASN KYYGDSVKGRFTISRDNSKNTLYLQMNSLRDEDTAVYYCARRFTHLRGQYIEDYGLDV WGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPRVYTLPPCRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLVS DGSFTLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG5G5GGGG5DIQM TQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSG SGSGTDFTLTISSLQPEDFATYYCQQGVSFPRTFGCGTKVEIKGGGGSGGGGSGGGGSGG GGSEVQLVESGGGLVKPGGSLRLSC AASGFTF S S YSMNWVRQAPGKCLEWVS SISS S S S YIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGAPIGAAAGWFDPWG QGTLVTVSS

[0470] AB-1424 / 1612-VH-CH1-CH2-CH3 (SEQ ID NO :272) (Chain “H”)EVQLVQSGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWYDASN KYYGDSVKGRFTISRDNSKNTLYLQMNSLRDEDTAVYYCARRFTHLRGQYIEDYGLDV WGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVCTLPPSRDELTENQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFLYSWLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0471] AB1424 / 1612-VL-CL (SEQ ID NO:273) (Chain “L”)EIVLTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSR FSGSGSGTDFTLTISSLQPEDFATYYCQQSYSIPLTFGGGTKVEIKRTVAAPSVFIFPPSDE QLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLS KADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0472] AB1424 / 1612-VH-CHl-CH2-CH3-A49MI-scFv (SEQ ID NO:271) represents the heavy chain portion of the Fab fragment, which comprises a heavy chain variable domain of BAFF-R-binding AB 1424 / 1612 (SEQ ID NO:250) and a CHI domain, connected to an Fc domain, further connected to an scFv. The scFv has the amino acid sequence of SEQ ID NO:275, which comprises a heavy chain variable domain of NKG2D-binding A49MI (SEQ ID NO:95) connected to the C-terminus of a light chain variable domain of A49MI (SEQ ID NO:85) via a (G4S)4 linker. The scFv also comprises substitution of Cys in the VH and VL regions at G44 and G100, facilitating formation of a disulfide bridge between the VH and VL of the scFv. The scFv of AB1424 / 1612-VH-CHl-CH2-CH3-A49MI-scFv is linked to the C-terminus of the CH3 domain by a short SGSGGGGS (SEQ ID NO:274) linker. The Fc domain in AB 1424 / 1612- VH- CHl-CH2-CH3-A49MI-scFv includes Q347R, D399V, and F405T substitutions for heterodimerization and an S354C substitution for forming a disulfide bond with a Y349C substitution in AB-1424 / 1612-VH-CH1-CH2-CH3 as described below.

[0473] AB1424 / 1612-VH-CH1-CH2-CH3 (SEQ ID NO:272) represents the heavy chain portion of the Fab fragment, which comprises a heavy chain variable domain of BAFF-R-binding AB1424 / 1612 (SEQ ID NO:250) and a CHI domain, connected to an Fc domain. The Fc domain in A49MI-VH-CH1-Fc includes a Y349C substitution in the CH3 domain, which forms a disulfide bond with an S354C substitution on the Fc in AB 1424 / 1612-VH-CH1-CH2-CH3 - A49MI-scFv. In AB1424 / 1612-VH-CH1-CH2-CH3, the Fc domain also includes K360E and K409W substitutions for heterodimerization with the Fc in AB 1424 / 1612- VH-CH1-CH2-CH3- A49MI-scFv.

[0474] AB1424 / 1612-VL-CL (SEQ ID NO:273) represents the light chain portion of theFab fragment comprising a light chain variable domain of BAFF-R-binding AB1424 / 1612 (SEQ ID NO:251) and a light chain constant domain.

[0475] In certain embodiments, an F3’ TriNKET described in the present disclosure is identical to one of the exemplary TriNKETs described above, except that (a) the Fc domain linked to the NKG2D-binding Fab fragment includes Q347R, D399V, and F405T substitutions in the CH3 domain for heterodimerization, and the Fc domain linked to the BAFF-R-binding scFv includes matching K360E and K409W substitution in the CH3 domain; and / or (b) the Fc domain linked to the NKG2D-binding Fab fragment includes an S354C substitution in the CH3 domain, and the Fc domain linked to the BAFF-R-binding scFv includes a matching Y349C substitution in the CH3 domain for forming a disulfide bond.

[0476] In certain embodiments, a 2-Fab TriNKET described in the present disclosure is identical to one of the exemplary TriNKETs described above, except that the Fc domain linked to the NKG2D-binding Fab fragment includes a F405L substitution in the CH3 domain for heterodimerization, and the Fc domain linked to the BAFF-R-binding Fab fragment includes a matching K409R substitution in the CH3 domain.

[0477] A skilled person in the art would appreciate that during production and / or storage of proteins, N-terminal glutamate (E) or glutamine (Q) can be cyclized to form a lactam (e.g., spontaneously or catalyzed by an enzyme present during production and / or storage).Accordingly, in some embodiments where the N-terminal residue of an amino acid sequence of a polypeptide is E or Q, a corresponding amino acid sequence with the E or Q replaced with pyroglutamate is also contemplated herein.

[0478] A skilled person in the art would also appreciate that during protein production and / or storage, the C-terminal lysine (K) of a protein can be removed (e.g., spontaneously or catalyzed by an enzyme present during production and / or storage). Such removal of K is often observed with proteins that comprise an Fc domain at its C-terminus. Accordingly, in some embodiments where the C-terminal residue of an amino acid sequence of a polypeptide (e.g., an Fc domain sequence) is K, a corresponding amino acid sequence with the K removed is also contemplated herein.

[0479] The multispecific proteins described above can be made using recombinant DNA technology well known to a skilled person in the art. For example, a first nucleic acid sequence encoding the first immunoglobulin heavy chain can be cloned into a first expression vector; a second nucleic acid sequence encoding the second immunoglobulin heavy chain can be clonedinto a second expression vector; a third nucleic acid sequence encoding the immunoglobulin light chain can be cloned into a third expression vector; and the first, second, and third expression vectors can be stably transfected together into host cells to produce the multimeric proteins.

[0480] To achieve the highest yield of the multispecific protein, different ratios of the first, second, and third expression vector can be explored to determine the optimal ratio for transfection into the host cells. After transfection, single clones can be isolated for cell bank generation using methods known in the art, such as limited dilution, ELISA, FACS, microscopy, or Clonepix.

[0481] Clones can be cultured under conditions suitable for bio-reactor scale-up and maintained expression of the multispecific protein. The multispecific proteins can be isolated and purified using methods known in the art including centrifugation, depth filtration, cell lysis, homogenization, freeze-thawing, affinity purification, gel filtration, ion exchange chromatography, hydrophobic interaction exchange chromatography, and mixed-mode chromatography.II. CHARACTERISTICS OF THE MULTISPECIFIC PROTEINS

[0482] The multispecific proteins described herein include an NKG2D-binding site, a BAFF-R binding site, and an antibody Fc domain or a portion thereof sufficient to bind CD 16, or an antigen-binding site that binds CD 16. In some embodiments, the multispecific proteins contains an additional antigen-binding site that binds BAFF-R, as exemplified in the F4- TriNKET format e.g., FIGs. 2C and 2D).

[0483] In some embodiments, the multispecific proteins display similar thermal stability to the corresponding monoclonal antibody, i.e., a monoclonal antibody containing the same BAFF- R binding site as the one incorporated in the multispecific proteins.

[0484] In some embodiments, the multispecific proteins simultaneously bind to cells expressing NKG2D and / or CD16, such as NK cells, and cells expressing BAFF-R, such as certain tumor cells. Binding of the multispecific proteins to NK cells can enhance the activity of the NK cells toward destruction of the BAFF-R expressing cells (e.g., BAFF-R expressing tumor cells). It has been reported that NK cells exhibit more potent cytotoxicity against target cells thatare stressed (see Chan et al., (2014) Cell Death Differ. 21(1):5-14). Without wishing to be bound by theory, it is hypothesized that when NK cells are engaged to a population of cells by a TriNKET, the NK cells may selectively kill the target cells that are stressed (e.g., malignant cells and cells in a tumor microenvironment). This mechanism could contribute to increased specificity and reduced toxicity of TriNKETs, making it possible to selectively clear the stressed cells even if expression of BAFF-R is not limited to the desired target cells.

[0485] In some embodiments, the multispecific proteins bind to BAFF-R with a similar affinity to the corresponding the anti-BAFF-R monoclonal antibody (i.e., a monoclonal antibody containing the same BAFF-R binding site as the one incorporated in the multispecific proteins). In some embodiments, the multispecific proteins are more effective in killing the tumor cells expressing BAFF-R than the corresponding monoclonal antibodies.

[0486] In certain embodiments, the multispecific proteins described herein, which include a binding site for BAFF-R, activate primary human NK cells when co-culturing with cells expressing BAFF-R. NK cell activation is marked by the increase in CD107a degranulation and IFN-y cytokine production. Furthermore, compared to a corresponding anti-BAFF-R monoclonal antibody, the multispecific proteins can show superior activation of human NK cells in the presence of cells expressing BAFF-R.

[0487] In some embodiments, the multispecific proteins described herein, which include a binding site for BAFF-R, enhance the activity of rested and IL-2-activated human NK cells when co-culturing with cells expressing BAFF-R.

[0488] In some embodiments, compared to the corresponding monoclonal antibody that binds to BAFF-R, the multispecific proteins offer an advantage in targeting tumor cells that express medium and low levels of BAFF-R.

[0489] In some embodiments, the bivalent F4 format of the TriNKETs i.e., TriNKETs include an additional antigen-binding site that binds to BAFF-R) improve the avidity with which the TriNKETs bind to BAFF-R, which in effect stabilizes expression and maintenance of high levels of BAFF-R on the surface of the tumor cells. In some embodiments, the F4-TriNKETs mediate more potent killing of tumor cells than the corresponding F3-TriNKETs or F3’- TriNKETs.III. THERAPEUTIC APPLICATIONS

[0490] The present application also describes methods for treating autoimmune disease or cancer using a multispecific binding protein described herein and / or a pharmaceutical composition described herein. The methods may be used to treat a variety of cancers or autoimmune diseases expressing BAFF-R.

[0491] The therapeutic method can be characterized according to the cancer to be treated. The cancer to be treated can be characterized according to the presence of a particular antigen expressed on the surface of the cancer cell, e.g., BAFF-R.

[0492] Cancers characterized by the expression of BAFF-R, include, without limitation, B- cell non-Hodgkin’s lymphoma (B-NHL), such as chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), marginal zone lymphoma, mucosa-associated lymphoid tissue (MALT) lymphoma, primary mediastinal B-cell lymphoma, acute lymphocytic leukemia (ALL); and autoimmune inflammatory diseases.

[0493] It is contemplated that the protein, conjugate, cells, and / or pharmaceutical compositions described in the present disclosure can be used to treat a variety of cancers, not limited to cancers in which the cancer cells or the cells in the cancer microenvironment express BAFF-R.

[0494] In certain embodiments, the cancer is a solid tumor. In certain other embodiments, the cancer is brain cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, colorectal cancer, endometrial cancer, esophageal cancer, leukemia, lung cancer, liver cancer, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer, renal cancer, stomach cancer, testicular cancer, or uterine cancer. In yet other embodiments, the cancer is a vascularized tumor, squamous cell carcinoma, adenocarcinoma, small cell carcinoma, melanoma, glioma, neuroblastoma, sarcoma e.g., an angiosarcoma or chondrosarcoma), larynx cancer, parotid cancer, biliary tract cancer, thyroid cancer, acral lentiginous melanoma, actinic keratoses, acute lymphocytic leukemia, acute myeloid leukemia, adenoid cystic carcinoma, adenomas, adenosarcoma, adenosquamous carcinoma, anal canal cancer, anal cancer, anorectum cancer, astrocytic tumor, Bartholin gland carcinoma, basal cell carcinoma, biliary cancer, bone cancer, bone marrow cancer, bronchial cancer, bronchial gland carcinoma, carcinoid,cholangiocarcinoma, chondrosarcoma, choroid plexus papilloma / carcinoma, chronic lymphocytic leukemia, chronic myeloid leukemia, clear cell carcinoma, connective tissue cancer, cystadenoma, digestive system cancer, duodenum cancer, endocrine system cancer, endodermal sinus tumor, endometrial hyperplasia, endometrial stromal sarcoma, endometrioid adenocarcinoma, endothelial cell cancer, ependymal cancer, epithelial cell cancer, Ewing's sarcoma, eye and orbit cancer, female genital cancer, focal nodular hyperplasia, gallbladder cancer, gastric antrum cancer, gastric fundus cancer, gastrinoma, glioblastoma, glucagonoma, heart cancer, hemangioblastomas, hemangioendothelioma, hemangiomas, hepatic adenoma, hepatic adenomatosis, hepatobiliary cancer, hepatocellular carcinoma, Hodgkin's disease, ileum cancer, insulinoma, intraepithelial neoplasia, intraepithelial squamous cell neoplasia, intrahepatic bile duct cancer, invasive squamous cell carcinomajejunum cancer oint cancer, Kaposi's sarcoma, pelvic cancer, large cell carcinoma, large intestine cancer, leiomyosarcoma, lentigo maligna melanomas, lymphoma, male genital cancer, malignant melanoma, malignant mesothelial tumors, medulloblastoma, medulloepithelioma, meningeal cancer, mesothelial cancer, metastatic carcinoma, mouth cancer, mucoepidermoid carcinoma, multiple myeloma, muscle cancer, nasal tract cancer, nervous system cancer, neuroepithelial adenocarcinoma nodular melanoma, non-epithelial skin cancer, non-Hodgkin's lymphoma, oat cell carcinoma, oligodendroglial cancer, oral cavity cancer, osteosarcoma, papillary serous adenocarcinoma, penile cancer, pharynx cancer, pituitary tumors, plasmacytoma, pseudosarcoma, pulmonary blastoma, rectal cancer, renal cell carcinoma, respiratory system cancer, retinoblastoma, rhabdomyosarcoma, sarcoma, serous carcinoma, sinus cancer, skin cancer, small cell carcinoma, small intestine cancer, smooth muscle cancer, soft tissue cancer, somatostatin-secreting tumor, spine cancer, squamous cell carcinoma, striated muscle cancer, submesothelial cancer, superficial spreading melanoma, T cell leukemia, tongue cancer, undifferentiated carcinoma, ureter cancer, urethra cancer, urinary bladder cancer, urinary system cancer, uterine cervix cancer, uterine corpus cancer, uveal melanoma, vaginal cancer, verrucous carcinoma, VIPoma, vulva cancer, well differentiated carcinoma, or Wilms tumor.

[0495] In certain embodiments, the cancer is a hematologic malignancy. In certain embodiments, the hematologic malignancy is leukemia. In certain embodiments, selected from the group consisting of acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), myelodysplasia, myelodysplastic syndromes, acute T-lymphoblastic leukemia, or acutepromyelocytic leukemia, chronic myelomonocytic leukemia, or myeloid blast crisis of chronic myeloid leukemia.

[0496] In some embodiments, the present application provides methods for treating an autoimmune inflammatory disease using a multispecific binding protein described herein and / or a pharmaceutical composition described herein. The methods may be used to treat a variety of BAFF-R-expressing B cell-associated autoimmune inflammatory diseases, including, without limitation, multiple sclerosis, systemic lupus erythematosus, Graves’ disease, Hashimoto’s thyroiditis, rheumatoid arthritis, inflammatory bowel disease, type I diabetes, Guillain-Barre syndrome, chronic inflammatory demyelinating polyneuropathy, psoriasis, myasthenia gravis, and vasculitis.IV. COMBINATION THERAPY

[0497] Another aspect of the present application provides for combination therapy. A multispecific binding protein described herein can be used in combination with additional therapeutic agents to treat autoimmune disease or to treat cancer.

[0498] Exemplary therapeutic agents that may be used as part of a combination therapy in treating autoimmune inflammatory diseases are described in Li et al. (2017) Front. Pharmacol., 8:460, and include, for example, non-steroidal anti-inflammatory drugs (NSAIDs) (e.g., COX-2 inhibitors), glucocorticoids (e.g., prednisone / prednisolone, methylprednisolone, and the fluorinated glucocorticoids such as dexamethasone and betamethasone), disease-modifying antirheumatic drugs (DMARDs) (e.g., methotrexate, leflunomide, gold compounds, sulfasalazine, azathioprine, cyclophosphamide, antimalarials, D-penicillamine, and cyclosporine), anti-TNF biologies (e.g., infliximab, etanercept, adalimumab, golimumab, Certolizumab pegol, and their biosimilars), and other biologies targeting CTLA-4 (e.g., abatacept), IL-6 receptor (e.g., tocilizumab), IL-1 (e.g., anakinra), Thl immune responses (IL- 12 / IL-23) (e.g., ustekinumab), Thl7 immune responses (IL-17) (e.g., secukinumab) and CD20 (e.g., rituximab).

[0499] Exemplary therapeutic agents that may be used as part of a combination therapy in treating cancer include, for example, radiation, mitomycin, tretinoin, ribomustin, gemcitabine, vincristine, etoposide, cladribine, mitobronitol, methotrexate, doxorubicin, carboquone, pentostatin, nitracrine, zinostatin, cetrorelix, letrozole, raltitrexed, daunorubicin, fadrozole,fotemustine, thymalfasin, sobuzoxane, nedaplatin, cytarabine, bicalutamide, vinorelbine, vesnarinone, aminoglutethimide, amsacrine, proglumide, elliptinium acetate, ketanserin, doxifluridine, etretinate, isotretinoin, streptozocin, nimustine, vindesine, flutamide, drogenil, butocin, carmofur, razoxane, sizofilan, carboplatin, mitolactol, tegafur, ifosfamide, prednimustine, picibanil, levamisole, teniposide, improsulfan, enocitabine, lisuride, oxymetholone, tamoxifen, progesterone, mepitiostane, epitiostanol, formestane, interferon-alpha, interferon-2 alpha, interferon-beta, interferon-gamma (IFN-y), colony stimulating factor- 1, colony stimulating factor-2, denileukin diftitox, interleukin-2, luteinizing hormone releasing factor and variations of the aforementioned agents that may exhibit differential binding to its cognate receptor, or increased or decreased serum half-life.

[0500] An additional class of agents that may be used as part of a combination therapy in treating cancer is immune checkpoint inhibitors. Exemplary immune checkpoint inhibitors include agents that inhibit one or more of (i) cytotoxic T lymphocyte-associated antigen 4 (CTLA4), (ii) programmed cell death protein 1 (PD1), (iii) PDL1, (iv) LAG3, (v) B7-H3, (vi) B7-H4, and (vii) TIM3. The CTLA4 inhibitor ipilimumab has been approved by the United States Food and Drug Administration for treating melanoma.

[0501] Yet other agents that may be used as part of a combination therapy in treating cancer are monoclonal antibody agents that target non-checkpoint targets (e.g., herceptin) and non-cytotoxic agents e.g., tyrosine-kinase inhibitors).

[0502] Yet other categories of anti-cancer agents include, for example: (i) an inhibitor selected from an ALK Inhibitor, an ATR Inhibitor, an A2A Antagonist, a Base Excision Repair Inhibitor, a Bcr-Abl Tyrosine Kinase Inhibitor, a Bruton's Tyrosine Kinase Inhibitor, a CDC7 Inhibitor, a CHK1 Inhibitor, a Cyclin-Dependent Kinase Inhibitor, a DNA-PK Inhibitor, an Inhibitor of both DNA-PK and mTOR, a DNMT1 Inhibitor, a DNMT1 Inhibitor plus 2-chloro- deoxyadenosine, an HD AC Inhibitor, a Hedgehog Signaling Pathway Inhibitor, an IDO Inhibitor, a JAK Inhibitor, a mTOR Inhibitor, a MEK Inhibitor, a MELK Inhibitor, a MTH1 Inhibitor, a PARP Inhibitor, a Phosphoinositide 3 -Kinase Inhibitor, an Inhibitor of both PARP1 and DHODH, a Proteasome Inhibitor, a Topoisomerase-II Inhibitor, a Tyrosine Kinase Inhibitor, a VEGFR Inhibitor, and a WEE1 Inhibitor; (ii) an agonist of 0X40, CD 137, CD40, GITR,CD27, HVEM, TNFRSF25, or ICOS; and (iii) a cytokine selected from IL-12, IL-15, GM-CSF, and G-CSF.

[0503] Proteins of the present application can also be used as an adjunct to surgical removal of the primary lesion.

[0504] The amount of multispecific binding protein and additional therapeutic agent, and the relative timing of administration, may be selected in order to achieve a desired combined therapeutic effect. For example, when administering a combination therapy to a patient in need of such administration, the therapeutic agents in the combination, or a pharmaceutical composition or compositions comprising the therapeutic agents, may be administered in any order such as, for example, sequentially, concurrently, together, simultaneously and the like. Further, for example, a multispecific binding protein may be administered during a time when the additional therapeutic agent(s) exerts its prophylactic or therapeutic effect, or vice versa.V. PHARMACEUTICAL COMPOSITIONS

[0505] The present disclosure also describes pharmaceutical compositions that contain a therapeutically effective amount of a protein described herein. The composition can be formulated for use in a variety of drug delivery systems. One or more physiologically acceptable excipients or carriers can also be included in the composition for proper formulation. Suitable formulations for use in the present disclosure are found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, Pa., 17th ed., 1985. For a brief review of methods for drug delivery, see, e.g., Langer (Science 249: 1527-1533, 1990).

[0506] The intravenous drug delivery formulation described in the present application may be contained in a bag, a pen, or a syringe. In certain embodiments, the bag may be connected to a channel comprising a tube and / or a needle. In certain embodiments, the formulation may be a lyophilized formulation or a liquid formulation. In certain embodiments, the formulation may be freeze-dried (lyophilized) and contained in about 12-60 vials. In certain embodiments, the formulation may be freeze-dried and 45 mg of the freeze-dried formulation may be contained in one vial. In certain embodiments, the about 40 mg to about 100 mg of freeze-dried formulation may be contained in one vial. In certain embodiments, freeze-dried formulation from 12, 27, or 45 vials are combined to obtain a therapeutic dose of the protein in the intravenous drug formulation. In certain embodiments, the formulation may be a liquid formulation and stored asabout 250 mg / vial to about 1000 mg / vial. In certain embodiments, the formulation may be a liquid formulation and stored as about 600 mg / vial. In certain embodiments, the formulation may be a liquid formulation and stored as about 250 mg / vial.

[0507] The protein could exist in a liquid aqueous pharmaceutical formulation including a therapeutically effective amount of the protein in a buffered solution forming a formulation.

[0508] These compositions may be sterilized by conventional sterilization techniques, or may be sterile filtered. The resulting aqueous solutions may be packaged for use as-is, or lyophilized, the lyophilized preparation being combined with a sterile aqueous carrier prior to administration. The pH of the preparations typically will be between 3 and 11, for example between 5 and 9 or between 6 and 8, and in certain embodiments, between 7 and 8, such as 7 to 7.5. The resulting compositions in solid form may be packaged in multiple single dose units, each containing a fixed amount of the above-mentioned agent or agents. The composition in solid form can also be packaged in a container for a flexible quantity.

[0509] In certain embodiments, the present application describes a formulation with an extended shelf life including a multispecific binding protein as described herein, in combination with mannitol, citric acid monohydrate, sodium citrate, disodium phosphate dihydrate, sodium dihydrogen phosphate dihydrate, sodium chloride, polysorbate 80, water, and sodium hydroxide.

[0510] In certain embodiments, an aqueous formulation is prepared including a protein of the present disclosure in a pH-buffered solution. The buffer of the formulation may have a pH ranging from about 4 to about 8, e.g., from about 4.5 to about 6.0, or from about 4.8 to about 5.5, or may have a pH of about 5.0 to about 5.2. Ranges intermediate to the above recited pH's are also intended to be part of this disclosure. For example, ranges of values using a combination of any of the above recited values as upper and / or lower limits are intended to be included.Examples of buffers that will control the pH within this range include acetate (e.g., sodium acetate), succinate (such as sodium succinate), gluconate, histidine, citrate and other organic acid buffers.

[0511] In certain embodiments, the formulation includes a buffer system which contains citrate and phosphate to maintain the pH in a range of about 4 to about 8. In certain embodiments the pH range may be from about 4.5 to about 6.0, or from about pH 4.8 to about 5.5, or in a pH range of about 5.0 to about 5.2. In certain embodiments, the buffer system includes citric acidmonohydrate, sodium citrate, di sodium phosphate dihydrate, and / or sodium dihydrogen phosphate dihydrate. In certain embodiments, the buffer system includes about 1.3 mg / mL of citric acid (e.g., 1.305 mg / mL), about 0.3 mg / mL of sodium citrate (e.g., 0.305 mg / mL), about 1.5 mg / mL of disodium phosphate dihydrate (e.g., 1.53 mg / mL), about 0.9 mg / mL of sodium dihydrogen phosphate dihydrate (e.g., 0.86 mg / mL), and about 6.2 mg / mL of sodium chloride (e.g., 6.165 mg / mL). In certain embodiments, the buffer system includes about 1 to about 1.5 mg / mL of citric acid, about 0.25 to about 0.5 mg / mL of sodium citrate, about 1.25 to about 1.75 mg / mL of disodium phosphate dihydrate, about 0.7 to about 1.1 mg / mL of sodium dihydrogen phosphate dihydrate, and about 6.0 to about 6.4 mg / mL of sodium chloride. In certain embodiments, the pH of the formulation is adjusted with sodium hydroxide.

[0512] A polyol, which acts as a tonicifier and may stabilize the antibody, may also be included in the formulation. The polyol is added to the formulation in an amount which may vary with respect to the desired isotonicity of the formulation. In certain embodiments, the aqueous formulation may be isotonic. The amount of polyol added may also be altered with respect to the molecular weight of the polyol. For example, a lower amount of a monosaccharide (e.g., mannitol) may be added, compared to a disaccharide (such as trehalose). In certain embodiments, the polyol which may be used in the formulation as a tonicity agent is mannitol. In certain embodiments, the mannitol concentration may be about 5 to about 20 mg / mL. In certain embodiments, the concentration of mannitol may be about 7.5 to about 15 mg / mL. In certain embodiments, the concentration of mannitol may be about 10 to about 14 mg / mL. In certain embodiments, the concentration of mannitol may be about 12 mg / mL. In certain embodiments, the polyol sorbitol may be included in the formulation.

[0513] A detergent or surfactant may also be added to the formulation. Exemplary detergents include nonionic detergents such as polysorbates (e.g., polysorbates 20, 80 etc.) or pol oxamers (e.g., pol oxamer 188). The amount of detergent added is such that it reduces aggregation of the formulated antibody and / or minimizes the formation of particulates in the formulation and / or reduces adsorption. In certain embodiments, the formulation may include a surfactant which is a polysorbate. In certain embodiments, the formulation may contain the detergent polysorbate 80 or Tween 80. Tween 80 is a term used to describe polyoxyethylene (20) sorbitanmonooleate (see Fiedler, Lexikon der Hifsstoffe, Editio Cantor Verlag Aulendorf, 4th ed., 1996). In certain embodiments, the formulation may contain between about 0.1 mg / mL andabout 10 mg / mL of polysorbate 80, or between about 0.5 mg / mL and about 5 mg / mL. In certain embodiments, about 0.1% polysorbate 80 may be added in the formulation.

[0514] In embodiments, a multispecific binding protein as described in the present application is formulated as a liquid formulation. The liquid formulation may be presented at a 10 mg / mL concentration in either a USP / Ph Eur type I 50R vial closed with a rubber stopper and sealed with an aluminum crimp seal closure. The stopper may be made of elastomer complying with USP and Ph Eur. In certain embodiments vials may be filled with 61.2 mL of the protein product solution in order to allow an extractable volume of 60 mL. In certain embodiments, the liquid formulation may be diluted with 0.9% saline solution.

[0515] In certain embodiments, the liquid formulation as described in this application may be prepared as a 10 mg / mL concentration solution in combination with a sugar at stabilizing levels. In certain embodiments the liquid formulation may be prepared in an aqueous carrier. In certain embodiments, a stabilizer may be added in an amount no greater than that which may result in a viscosity undesirable or unsuitable for intravenous administration. In certain embodiments, the sugar may be a disaccharide, e.g., sucrose. In certain embodiments, the liquid formulation may also include one or more of a buffering agent, a surfactant, and a preservative.

[0516] In certain embodiments, the pH of the liquid formulation may be set by addition of a pharmaceutically acceptable acid and / or base. In certain embodiments, the pharmaceutically acceptable acid may be hydrochloric acid. In certain embodiments, the base may be sodium hydroxide.

[0517] In addition to aggregation, deamidation is a common product variant of peptides and proteins that may occur during fermentation, harvest / cell clarification, purification, drug substance / drug product storage and during sample analysis. Deamidation is the loss of NH3 from a protein forming a succinimide intermediate that can undergo hydrolysis. The succinimide intermediate results in a 17 dalton mass decrease of the parent peptide. The subsequent hydrolysis results in an 18 dalton mass increase. Isolation of the succinimide intermediate is difficult due to instability under aqueous conditions. As such, deamidation is typically detectable as 1 dalton mass increase. Deamidation of an asparagine results in either aspartic or isoaspartic acid. The parameters affecting the rate of deamidation include pH, temperature, solvent dielectric constant, ionic strength, primary sequence, local polypeptide conformation and tertiary structure.The amino acid residues adjacent to Asn in the peptide chain affect deamidation rates. Gly and Ser following an Asn in protein sequences results in a higher susceptibility to deamidation.

[0518] In certain embodiments, the liquid formulation as described in this application may be preserved under conditions of pH and humidity to prevent deamination of the protein product.

[0519] The aqueous carrier of interest herein is one which is pharmaceutically acceptable (safe and non-toxic for administration to a human) and is useful for the preparation of a liquid formulation. Illustrative carriers include sterile water for injection (SWFI), bacteriostatic water for injection (BWFI), a pH buffered solution (e.g., phosphate-buffered saline), sterile saline solution, Ringer's solution or dextrose solution.

[0520] A preservative may be optionally added to the formulations described herein to reduce bacterial action. The addition of a preservative may, for example, facilitate the production of a multi-use (multiple-dose) formulation.

[0521] Intravenous (IV) formulations may be an administration route in particular instances, such as when a patient is in the hospital after transplantation receiving all drugs via the IV route. In certain embodiments, the liquid formulation is diluted with 0.9% Sodium Chloride solution before administration. In certain embodiments, the diluted drug product for injection is isotonic and suitable for administration by intravenous infusion.

[0522] In certain embodiments, a salt or buffer components may be added in an amount of 10 mM - 200 mM. The salts and / or buffers are pharmaceutically acceptable and are derived from various known acids (inorganic and organic) with “base forming” metals or amines. In certain embodiments, the buffer may be phosphate buffer. In certain embodiments, the buffer may be glycinate, carbonate, citrate buffers, in which case, sodium, potassium or ammonium ions can serve as counterion.

[0523] A multispecific binding protein as described in the present application could exist in a lyophilized formulation including the proteins and a lyoprotectant. The lyoprotectant may be a sugar, e.g., a disaccharide. In certain embodiments, the lyoprotectant may be sucrose or maltose. The lyophilized formulation may also include one or more of a buffering agent, a surfactant, a bulking agent, and / or a preservative.

[0524] The amount of sucrose or maltose useful for stabilization of the lyophilized drug product may be in a weight ratio of at least 1 :2 protein to sucrose or maltose. In certain embodiments, the protein to sucrose or maltose weight ratio may be of from 1 :2 to 1 :5.

[0525] In certain embodiments, the pH of the formulation, prior to lyophilization, may be set by addition of a pharmaceutically acceptable acid and / or base. In certain embodiments the pharmaceutically acceptable acid may be hydrochloric acid. In certain embodiments, the pharmaceutically acceptable base may be sodium hydroxide.

[0526] Before lyophilization, the pH of the solution containing a protein of the present disclosure may be adjusted between 6 to 8. In certain embodiments, the pH range for the lyophilized drug product may be from 7 to 8.

[0527] In certain embodiments, a salt or buffer components may be added in an amount of 10 mM - 200 mM. The salts and / or buffers are pharmaceutically acceptable and are derived from various known acids (inorganic and organic) with “base forming” metals or amines. In certain embodiments, the buffer may be phosphate buffer. In certain embodiments, the buffer may be glycinate, carbonate, citrate buffers, in which case, sodium, potassium or ammonium ions can serve as counterion.

[0528] In certain embodiments, a “bulking agent” may be added. A “bulking agent” is a compound which adds mass to a lyophilized mixture and contributes to the physical structure of the lyophilized cake (e.g., facilitates the production of an essentially uniform lyophilized cake which maintains an open pore structure). Illustrative bulking agents include mannitol, glycine, polyethylene glycol and sorbitol. The lyophilized formulations of the multispecific binding proteins described in the present application may contain such bulking agents.

[0529] A preservative may be optionally added to the formulations herein to reduce bacterial action. The addition of a preservative may, for example, facilitate the production of a multi-use (multiple-dose) formulation.

[0530] In certain embodiments, the lyophilized drug product may be constituted with an aqueous carrier. The aqueous carrier of interest herein is one which is pharmaceutically acceptable (e.g., safe and non-toxic for administration to a human) and is useful for the preparation of a liquid formulation, after lyophilization. Illustrative diluents include sterile waterfor injection (SWFI), bacteriostatic water for injection (BWFI), a pH buffered solution (e.g., phosphate-buffered saline), sterile saline solution, Ringer's solution or dextrose solution.

[0531] In certain embodiments, the lyophilized drug product is reconstituted with either Sterile Water for Injection, USP (SWFI) or 0.9% Sodium Chloride Injection, USP. During reconstitution, the lyophilized powder dissolves into a solution.

[0532] In certain embodiments, the lyophilized protein product is constituted to about 4.5 mL water for injection and diluted with 0.9% saline solution (sodium chloride solution).

[0533] Actual dosage levels of the active ingredients in the pharmaceutical compositions of multispecific binding proteins described in this application may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.

[0534] The specific dose can be a uniform dose for each patient, for example, 50-5000 mg of protein. Alternatively, a patient’s dose can be tailored to the approximate body weight or surface area of the patient. Other factors in determining the appropriate dosage can include the disease or condition to be treated or prevented, the severity of the disease, the route of administration, and the age, sex and medical condition of the patient. Further refinement of the calculations necessary to determine the appropriate dosage for treatment is routinely made by those skilled in the art, especially in light of the dosage information and assays disclosed herein. The dosage can also be determined through the use of known assays for determining dosages used in conjunction with appropriate dose-response data. An individual patient's dosage can be adjusted as the progress of the disease is monitored. Blood levels of the targetable construct or complex in a patient can be measured to see if the dosage needs to be adjusted to reach or maintain an effective concentration. Pharmacogenomics may be used to determine which targetable constructs and / or complexes, and dosages thereof, are most likely to be effective for a given individual (Schmitz et al., Clinica Chimica Acta 308: 43-53, 2001; Steimer el al., Clinica Chimica Acta 308: 33-41, 2001).

[0535] In general, dosages based on body weight are from about 0.01 pg to about 100 mg per kg of body weight, such as about 0.01 pg to about 100 mg / kg of body weight, about 0.01 pg to about 50 mg / kg of body weight, about 0.01 pg to about 10 mg / kg of body weight, about 0.01 pg to about 1 mg / kg of body weight, about 0.01 pg to about 100 pg / kg of body weight, about0.01 pg to about 50 pg / kg of body weight, about 0.01 pg to about 10 pg / kg of body weight, about 0.01 gg to about 1 gg / kg of body weight, about 0.01 gg to about 0.1 gg / kg of body weight, about 0.1 gg to about 100 mg / kg of body weight, about 0.1 gg to about 50 mg / kg of body weight, about 0.1 gg to about 10 mg / kg of body weight, about 0.1 gg to about 1 mg / kg of body weight, about 0.1 gg to about 100 gg / kg of body weight, about 0.1 gg to about 10 gg / kg of body weight, about 0.1 gg to about 1 gg / kg of body weight, about 1 gg to about 100 mg / kg of body weight, about 1 gg to about 50 mg / kg of body weight, about 1 gg to about 10 mg / kg of body weight, about 1 gg to about 1 mg / kg of body weight, about 1 gg to about 100 gg / kg of body weight, about 1 gg to about 50 gg / kg of body weight, about 1 gg to about 10 gg / kg of body weight, about 10 gg to about 100 mg / kg of body weight, about 10 gg to about 50 mg / kg of body weight, about 10 gg to about 10 mg / kg of body weight, about 10 gg to about 1 mg / kg of body weight, about 10 gg to about 100 gg / kg of body weight, about 10 gg to about 50 gg / kg of body weight, about 50 gg to about 100 mg / kg of body weight, about 50 gg to about 50 mg / kg of body weight, about 50 gg to about 10 mg / kg of body weight, about 50 gg to about 1 mg / kg of body weight, about 50 gg to about 100 gg / kg of body weight, about 100 gg to about 100 mg / kg of body weight, about 100 gg to about 50 mg / kg of body weight, about 100 gg to about 10 mg / kg of body weight, about 100 gg to about 1 mg / kg of body weight, about 1 mg to about 100 mg / kg of body weight, about 1 mg to about 50 mg / kg of body weight, about 1 mg to about 10 mg / kg of body weight, about 10 mg to about 100 mg / kg of body weight, about 10 mg to about 50 mg / kg of body weight, about 50 mg to about 100 mg / kg of body weight.

[0536] Doses may be given once or more times daily, weekly, monthly or yearly, or even once every 2 to 20 years. Persons of ordinary skill in the art can easily estimate repetition rates for dosing based on measured residence times and concentrations of the targetable construct or complex in bodily fluids or tissues. Administration of the multispecific binding proteins described in the present application could be intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, intrapleural, intrathecal, intracavitary, by perfusion through a catheter or by direct intralesional injection. This may be administered once or more times daily, once or more times weekly, once or more times monthly, and once or more times annually.

[0537] The description above provides multiple aspects and embodiments of themultispecific binding proteins described in the application. The patent application specifically contemplates all combinations and permutations of the aspects and embodiments. The use of any and all examples, or exemplary language herein, for example, “such as” or “including,” is intended merely to illustrate better the multispecific binding proteins described in the present application, and does not pose a limitation on the scope of the disclosure, unless so expressly stated. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the multispecific binding proteins described in the present application.EXAMPLES

[0538] The following examples are merely illustrative and are not intended to limit the scope or content of the multispecific binding proteins described in the present application in any way.Example 1 - Assessment of TriNKET binding to cell expressed human BAFF-R

[0539] The BAFF-R positive human B lymphoblastoid RAJI cell line was used to assess TriNKET binding to cell surface BAFF-R. Certain BAFF-R TriNKETs in the 2-Fab and F3’ formats, as described in the “exemplary multispecific binding proteins” subsection above, were diluted and incubated with Raji cells. Binding patterns of TriNKETs and parental monoclonal antibodies were detected using a fluorophore conjugated anti-human IgG secondary antibody. The cells were then incubated with a fluorophore conjugated anti-human IgG secondary antibody and were analyzed by flow cytometry. The mean fluorescence intensity (MFI) values were normalized to secondary antibody only controls to obtain fold over background (FOB) values.

[0540] As shown in FIG. 18A-FIG. 18C, BAFF-R TriNKETs containing a BAFF-R binding site derived from hCOH-2 (FIG. 18A), Hu9.1-73 (FIG. 18B), and ianalumab-based antigen-binding site (the three versions, F3’, 2-Fab, and ianalumab-mAb, do not contain antibody-dependent cellular cytotoxicity-enhancing mutations present in the commercial ianalumab antibody) (FIG. 18C) bind with subnanomolar concentration and with similar or higher maximum MFI than the corresponding parental control antibodies, which does not contain ADCC-enhancing mutations used in ianalumab. The ECso values of these TriNKETs bindingBAFF-R are shown in Table 11. Similar results were obtained with a second BAFF-R positive cell line, Ramos (data not shown).Table 11. ECso values in BAFF-R binding assay using RAJI cells*The ianalumab constructs do not include antibody-dependent cellular cytotoxicity-enhancing mutations present in the commercial ianalumab antibody.Example 2 - Human NK cell cytotoxicity assay

[0541] Lysis of BAFF-R-expressing target cells by immune effector cells in the presence of the TriNKETs was measured by the DELFIA cytotoxicity assay. Briefly, human cancer cell line RAJI expressing BAFF-R was harvested from culture, washed with HBS, and resuspended in growth media at 106 / mL for labeling with BATDA reagent (Perkin Elmer ADO 116).Manufacturer instructions were followed for labeling of the target cells. After labeling, cells were washed three times with HBS, and were resuspended at 0.5-1.0xl05 / mL in culture media. 100 pl of BATDA labeled cells were added to each well of the 96-well plate. Monoclonal antibodies or TriNKETs against BAFF-R were diluted in culture media, and 50 pl of diluted mAb or TriNKET were added to each well.

[0542] To prepare NK cells, PBMCs were isolated from human peripheral blood buffy coats using density gradient centrifugation, washed, and prepared for NK cell isolation. NK cells were isolated using a negative selection technique with magnetic beads. Purity of isolated NKcells was typically >90% CD3'CD56+. Isolated NK cells were rested overnight and harvested from culture. The cells were then washed and resuspended at concentrations of 105-2.0xl06 / mL in culture media for an effector-to-target (E:T) ratio of 5: 1. 50 pl of NK cells were added to each well of the plate for a total of 200 pl culture volume. The plate was incubated at 37 °C with 5% CO2 for 2-3 hours.

[0543] After the incubation, the plate was removed from the incubator and the cells were pelleted by centrifugation at 200x g for 5 minutes. 20 pl of culture supernatant were transferred to a clean microplate and 200 pl of room temperature europium solution (Perkin Elmer C135- 100) were added to each well. The plate was protected from light and incubated on a plate shaker at 250 rpm for 15 minutes, then read using SpectraMax i3X instruments.

[0544] Spontaneous release of substance that can form a fluorescent chelate with europium was measured in target cells incubated in the absence of NK cells. Maximum release of such substance was measured in target cells lysed with 1% Triton-X. % Specific lysis was calculated as follows:% Specific lysis = ((Experimental release - Spontaneous release) / (Maximum release - Spontaneous release)) x 100%.

[0545] FIG. 19A-FIG. 19C show NK cell-mediated lysis of BAFF-R-positive RAJI cells by primary NK cells in the presence of BAFF-R-targeting TriNKETs derived from hCOH-2 (FIG. 19A), Genentech Hu9.1-73 (FIG. 19B), and ianalumab-based antigen-binding site (the three versions, F3’, 2-Fab, and ianalumab-mAb, do not contain antibody-dependent cellular cytotoxicity-enhancing mutations present in the commercial ianalumab antibody) (FIG. 19C). Parental BAFF-R targeted monoclonal antibodies showed little enhancement of NK cell mediated lysis of RAJI target cells. All BAFF-R targeted TriNKETs (hCOH-2-F3’, hCOH-2-2- Fab, Hu9.1-73-F3’, Hu9.1-73-2 -Fab, ianalumab-F3’, and ianalumab-2-Fab) showed superior lysis of target cells compared to the respective BAFF-R targeted monoclonal antibody. ECso values are shown in Table 12.Table 12. Potency of BAFF-R TriNKETs in comparison with parental mAb in primary NK mediated cytotoxicity assay.*Ianalumab constructs do not include antibody-dependent cellular cytotoxicity-enhancing mutations present in the commercial ianalumab antibody.

[0546] To confirm the cytoxicity findings, an NK cell line, KHYG-l-CD16aV was engineered to stably express CD16aV and NKG2D, and the assay was conducted as described above. Cytotoxicity was measured for TriNKETs and parental mAbs derived from hCOH-2 (FIG. 20A), Hu9.1-73 (FIG. 20B) and ianalumab-based antigen-binding site (the three versions, F3’, 2-Fab, and ianalumab-mAb, do not contain antibody-dependent cellular cytotoxicityenhancing mutations present in the commercial ianalumab antibody) (FIG. 20C). The ECso values and maximum lysis values were derived from the cell lysis curves by the GraphPad Prizm software using four parameter logistic non-linear regression curve fitting model (Table 13). All BAFF-R TriNKETs tested showed subnanomolar EC50 and high efficiency maximum lysis in the KHYG-1 CD16aV mediated cytotoxicity assay. Similar results were obtained with a second BAFF-R positive cell line, Ramos (data not shown).Table 13. Potency of BAFF-R TriNKETs in comparison with parental mAb in primary KHYG- 1 CD16aV mediated cytotoxicity assay.*Ianalumab constructs do not include antibody-dependent cellular cytotoxicity-enhancing mutations present in the commercial ianalumab antibody.Example 3 - Generation and characterization of BAFF-R binding mAbsRecombinant protein immunization methods

[0547] BAFF-R-specific antibodies were generated by immunizing four different strains of mice (H2L2, NZBW, BALB-C, and SJL / J) with hBAFF-R-hFc-His fusion protein. Based on antisera titers, a total of seven mice from across the four different strains were selected for hybridoma fusion. Splenocytes from a subset of mice from each immunization arm were reserved for immune library generation; however, only splenocytes from H2L2 mice were used for yeast display mAb discovery.

[0548] From five mice fusions (splenocytes from two mice were pooled for H2L2 fusion and splenocytes from two mice were pooled for SJL / J fusion), sixteen 96-well plates per hybridoma fusion were analyzed by specificity ELISA, in which binding to human and cynomolgus monkey BAFF-R-hFc-His and binding to irrelevant-hFc-His protein was compared. Supernatants from 33 BAFF-R positive and specific hybridomas were selected for further analysis. Supernatants were tested for binding to BAFF-R+ isogenic CHO cells, and 16 positive hybridomas were further subcloned. Supernatants from the subclones were analyzed by specificity ELISA as described above and 20 BAFF-R positive and specific subclones were tested for binding to BAFF-R+ cells. Nine subclone mAbs demonstrated strong binding to BAFF-R+ cells and were sequenced. Six unique sequences were obtained, and the correspondingmAbs were further analyzed for their ability to block BAFF-R-BAFF interactions in a cell-based assay.

[0549] Binding of biotinylated BAFF to BAFF-R+ CHO cells was tested in the absence or presence of the six BAFF-R specific mAbs or an isotype control mAb. Reduction of mean fluorescent intensity (MFI) in the presence of antibody suggested that a mAb inhibited engagement of BAFF binding to BAFF-R, thus was designated as blocking antibody. All clones tested did not inhibit BAFF binding to BAFF-R+ cells, and therefore, all six were termed nonblocking (FIG. 21).DNA immunization methods

[0550] DNA immunization of two groups of SWR / J mice each was performed. One group was immunized with a full-length human BAFF-R cDNA construct, and the other with a mixture of full-length human BAFF-R and human BAFF-R extracellular domain cDNA constructs. Based on antisera titers, mice were pooled, and subsequently selected for single B cell sorting and another pool used for hybridoma fusion.

[0551] Single B cell sorting efforts yielded 44 human and cynomolgus monkey cross- reactive clones. These clones were sequenced, transiently expressed in 293 cells, and the specificity of the purified mAb was analyzed by flow cytometry in which binding to hBAFF-R+, cynoBAFF-R+isogenic CHO cells and to parental cell line was compared. Eight binders were purified and further analyzed for their ability to bind to BAFF-R and to block BAFF-R-BAFF interactions. All eight clones were determined non-blocking and demonstrated weak affinity for hBAFF-R+cancer cells.

[0552] Specificity of clones obtained by the traditional hybridoma approach analyzed by flow cytometry. The following assessment was performed: a) binding to cells expressing either full-length human BAFF-R or human BAFF-R extracellular domain was compared to binding to non-transfected parental cells; b) binding to hBAFF-R+and cynoBAFF-R+isogenic cell was compared to binding to the parental cells; c) binding to hBAFF-R+cancer cells. 25 positive hybridoma fusions were identified and based on the binding intensity 14 hybridoma fusions were sequenced. Five unique sequences were obtained and analyzed for their ability to bind BAFF-R+cells and block BAFF-R-BAFF interactions. Though all five clones were determined non-blocking clones (FIG. 22), four out of five clones (clones 3A1, 1B3-A7, 7G4 and 10H7-C5) demonstrated good affinity for hBAFF-R.BAFF-R specific scFvs discovered from yeast libraries

[0553] Yeast display was used to build scFv libraries from the splenocytes obtained from humanized H2L2 mice immunized with recombinant human hBAFF-R-hFc-His protein as described above. Three rounds of selection were carried out with biotinylated hBAFF-R-hFc- His at 5 nM. Individual yeast colonies were picked, sequenced, and sequences analyzed. Sequence convergence indicated the selection process was successful in enriching for binders and was therefore complete. Unique sequences were selected for further characterization. Three BAFF-R specific scFvs were discovered from one library (Table 14). However, these sequences were very similar to each other, and therefore only sequence 1129 A01 (also referred to as AB0369scFv) was selected for further study.Table 14. CDR sequences of BAFF-R binders discovered from yeast library

[0554] Flow cytometry was used to assess specificity of binding of AB0369scFv to hBAFF-R-hFc-His, hBAFF-R-GST-His, and negative control proteins with hFc tag or GST tag while displayed on yeast. AB0369scFv demonstrated medium to weak affinity towards hBAFF-R; however, it did not show binding to the negative control, thus suggesting high specificity for BAFF-R (FIG. 23).

[0555] 1129 A01 (AB0369 scFv) was converted into a multispecific binding protein comprising the scFv, and two non-BAFF-R binders, to yield AB0369. AB0369 was further analyzed for its abilities to bind to human (hBAFF-R-CHO) and cynomolgus monkey (cBAFF- R-CHO) BAFF-R+cells (FIG. 24A, FIG. 24B), lack non-specific interactions by polyspecificity reagent (PSR) assay (FIG. 25A-FIG. 25G), lyse BAFF-R+Ramos cancer cells (FIG. 26 and Table 15) and block BAFF-BAFF-R interactions (FIG. 27). AB0369 bound to both human and cynomolgus monkey BAFF-R on the surface of isogenic CHO cells, and BAFF-R binding was with ECso about 10 nM, making it a good choice for further development.Table 15. Potency of AB0369 in KHYG-l-CD16aV cytotoxicity assay.

[0556] The ability of AB0369 to block BAFF-R-BAFF interactions was tested in a cellbased blocking assay. Briefly, CHO cells expressing human BAFF-R were harvested, washed in cold FACS buffer, and seeded at a density of 100,000 cells per well. Test articles were diluted in FACS buffer, and 50 pL of diluted multispecific binding protein or mAb was added to cells, incubated on ice for 60 minutes, then washed with FACS buffer. 12 nM BAFF-biotin was diluted into FACS buffer, and 100 pL was added per well, incubated for 60 minutes on ice, then washed with FACS buffer. Cells were incubated with 100 pL of 1 :200 streptavidin-PE diluted in FACS buffer and incubated on ice for 30 minutes then washed with FACS buffer. Cells were then incubated in 100 pL of 1 : 1,000 dilution of live / dead dye in PBS for 15 minutes, then washed with FACS buffer, and fixed. After incubation, cells were washed with FACS buffer and resuspended in FACS buffer for analysis with flow cytometry. Median fluorescent intensity (MFI) of each sample and the secondary-only control was calculated. Maximum MFI was calculated as BAFF-biotin alone, and minimum MFI was calculated as streptavidin- Phycoerythrin alone. Data were fit to a four-parameter non-linear regression curve using GraphPad Prism.

[0557] These studies revealed that AB0369 was able to partially block BAFF-R-BAFF interactions. However, the blocking was significantly less potent than the ianalumab-based benchmark control, which does not contain antibody-dependent cellular cytotoxicity enhancing mutations unlike the parent antibody, presumably due to the low affinity of AB0369 (FIG. 27 and Table 16). As the AB0369 scFv was the only blocking antibody identified from all discovery efforts described above, it underwent further development by affinity maturation of CDRH3 and CDRH1 / CDRH2, as well as further amino acid changes to facilitate protein production and stability.Table 16. Summary of AB0369 and benchmark mAb blocking of BAFF binding to cellular BAFF-R.Affinity maturation of AB0369CDRH3 focused randomized affinity maturation

[0558] As described above, AB0369 demonstrated specific binding to BAFF-R expressing cells. To search for variants with improved binding affinities, a yeast display affinity maturation library was created by mutating the CDRH3 residue (RFTMLRGLIIEDYGMDV (SEQ ID NO:216)) of AB0369. To enrich for scFvs that have higher affinity towards hBAFF-R, two rounds of selection were carried out with biotinylated hBAFF-R-hFc-His at 1 nM (FIG. 28A- FIG. 28D). The affinities between the parental clone AB0369 and representative individual library clones were compared. Three rounds of FACS sorting resulted in nine clones that contained one or two amino acid differences (bolded) as compared to the parental clone (RFTMLRGWYIEDYGMDV (SEQ ID NO:224); RFTMLRGQYIEDYGMDV (SEQ ID NO:223); RFTMLRGWIIEDYGMDV (SEQ ID NO:225)) and exhibited higher binding affinity for hBAFF-R than the parental clone and parental-derived scFv, using an ianalumab-based scFv as a benchmark control (FIG. 29A-FIG. 29D).

[0559] The scFvs with highest hBAFF-R binding affinity were converted into multispecific binding proteins comprising the scFv and two non-BAFF-R binders, expressed in Expi293 cells, and further analyzed for their ability to bind to BAFF-R expressing cells (FIG. 10A) and ability to lyse BAFF-R expressing Ramos cancer cells (FIG. 30B, FIG. 30C). All multispecific binding proteins scored negatively in a poly-specificity assay, suggesting that the improved binding affinity was BAFF-R specific (FIG. 31A-FIG. 31E). Further studies demonstrated greater than three-fold improvement in BAFF-R binding, which translated into six- to ten-fold improvement in potency as measured by ECso (Table 17). Maximum lysis remained unchanged, suggesting that the improvement in BAFF-R binding affinity was the key driver of this improvement in potency.Table 17. Summary of cell binding and cytolysis demonstrated by multispecific binding proteins based on HCDR3 affinity matured variants compared to parental AB0369.CDRH1 and CDRH2 focused combinatory affinity maturation

[0560] Outcomes from the CDRH3 -focused affinity maturation studies demonstrated an improvement in affinity, and further improvement was highly desirable. Thus, the CDRH1 and CDRH2 sequences were selected for affinity maturation (CDRH1 : GFTFSSY (SEQ ID NO:214) and CDRH2: WYDGSN (SEQ ID NO:215)) using the matured CDRH3 backbone. The goal was to engineer and select binders with improved affinity over the parental clone (AB0369 scFv) or the CDRH3 optimized variants described above. This created a library with a randomized CDRH1 and CDRH2 while retaining an optimized CDRH3. Two rounds of FACS were performed to enrich for high-affinity binders (FIG. 32A-FIG. 32C).

[0561] After FACS, 24 clones were identified. It was observed that several clones with changes in CDRH1 (RFTMLRGWYIEDYGMDV (SEQ ID NO: 224);RFTMLRGQYIEDYGMDV (SEQ ID NO:223)); RFTMLRGWIIEDYGMDV(SEQ ID NO:225)) on the optimized CDRH3 backbone showed a significant improvement in hBAFF-R affinity compared to parental AB0369scFv (1129 A01) (FIG. 33A-FIG. 33D) or to an ianalumab-based scFv benchmark control (the scFv includes a VH and a VL that are based on the VH and VL sequences of ianalumab , but does not contain ADCC-enhancing mutations used in the parent antibody.) (FIG. 33E).

[0562] The scFvs with the highest hBAFF-R binding affinities were converted into multispecific binding proteins comprising the scFv and two non-BAFF-R binders, expressed in Expi293 cells, and further analyzed for their ability to bind to human BAFF-R expressing cells (FIG. 34 A), to bind to cynomolgus BAFF-R+cells (FIG. 34B), and to inhibit BAFF-R-BAFF interactions (FIG. 34C and Table 18). Tested multispecific binding proteins showed improvement in all three of these criteria and demonstrated efficient killing of BAFF-R+BJAB cells in a KHYG-l-CD16a-mediated cytotoxicity assay (FIG. 35, Table 19).Table 18. Summary of BAFF-R cell binding and BAFF-R-BAFF blocking demonstrated by multispecific binding proteins based on CDRH1 and CDRH2 affinity maturationTable 19. Potency of representative multispecific binding proteins based on CDRH1 and CDRH2 affinity maturation in a KHYG-1-CD16V cytolysis assay.Remediation of potential sequence liabilities

[0563] Because the affinity-matured clones contained amino acids in their CDRs that could negatively impact protein expression, stability, or immunogenicity, additional libraries were constructed to select for clones without these amino acids. Three rounds of selection were performed with 1 nM biotinylated hBAFF-R-hFc-His protein leading to enrichment of high affinity binders (FIG. 36A-FIG. 36D). 23 binders were identified altogether, 12 of which were predicted to be free of undesirable amino acids (“liability-corrected”).

[0564] Preferred clones from these libraries included AB0898, (the liability-corrected version of AB0682 described above), AB0899, and AB0900, which were successfully identified and tested for their binding to hBAFF-R while displayed on yeast. All clones showed higher affinity towards hBAFF-R than the parent, AB0369scFv (FIG. 37A-FIG. 37F).Characterization of liability-corrected multispecific binding proteins

[0565] Three of the liability-corrected clones were converted into multispecific binding proteins comprising the scFv and two non-BAFF-R binders, expressed in Expi293 cells, purified by a two-step purification process and characterized by size-exclusion chromatography (SEC), differential scanning calorimetry (DSC), binding to BAFF-R-expressing cells, and ability to lyse BJAB cells in a KHYG-l-CD16aV-mediated cytotoxicity assay. Characterization of these clones is summarized in Table 20 and demonstrates that the liability correction was successful. No negative effect on cell binding was observed and all three clones demonstrated potent killing of BAFF-R-expressing tumor cells (FIG. 38). However, the thermostability of the molecules was Tmi > 65 °C, as shown in FIG. 39A-FIG. 39C.Table 20. Summary of characterization of multispecific binding proteins expressing sequence liability corrected BAFF-R binders.

[0566] As described above, replacement of the potential sequence liability residues with certain amino acids in CDRs had minimal effect on binding affinity; however, BAFF-Rexpressing cell binding and thermostability data suggested that further improvement was desirable. Thus, the CDRH1 and CDRH2 sequences (CDRH1 : GFTFSSY (SEQ ID NO:214) and CDRH2: WYDGSN (SEQ ID NO:215)) were affinity matured into the liability-corrected CDRH3 backbone, and off-rate pressure was applied to select high affinity clones. Briefly, clones were preincubated with biotinylated hBAFF-R-hFc-His at 100 pM concentration, and then challenged with 1 pM non-biotinylated hBAFF-R-hFc-His for 2 hours. Yeast displaying anti- BAFF-R scFvs that remained bound to biotinylated hBAFF-R-hFc-His were sorted and the process was repeated three times to enrich for high affinity binders with slower off-rate. As shown in FIG. 40, clones remained bound to biotinylated hBAFF-R-hFc-His even after the off- rate pressure challenge, whereas the ianalumab-based scFv benchmark control lost binding to biotinylated hBAFF-R-hFc-His under these conditions, suggesting a slower dissociation rate.

[0567] Analysis of individual clones demonstrated high affinity towards hBAFF-R-hFc-His (FIG. 41) and importantly, the clones remained bound to biotinylated hBAFF-R-hFc-His.Notably, ianalumab-based benchmark scFv exhibited loss of binding to biotinylated hBAFF-R- hFc-His after the challenge (FIG. 41A and FIG. 41B). Several of the clones were eliminated from further consideration because they contained additional undesirable amino acids or properties. Sequences of selected clones from the above studies are shown in Table 21.Table 21. CDR sequences of selected clones.Potential sequence liabilities are bold-underlined and residues demonstrating diversity between clones are bolded.

[0568] Selected clones from the off-rate challenge studies described above were produced as multispecific binding proteins comprising an scFv of the respective binders and two non- BAFF-R binders, expressed in Expi293 cells, and characterized by binding to hBAFF-Rexpressing cells and cynomolgus BAFF-R expressing cells, ability to lyse BAFF-R expressing cancer cells in a KHYG-l-CD16aV-mediated cytotoxicity assay, ability to block BAFF-BAFF-R interactions, thermostability (differential scanning fluorimetry, DSF) and hydrophobicity (HIC) (results are summarized in Table 22). Binding affinity of AB 1080, AB 1081, and AB 1085 to BAFF-R+cells was improved as compared to the parental clones (FIG. 42A and FIG. 42B as compared to Table 20). Additionally, binding affinity to cynoBAFF-R was similar to binding affinity to hBAFF-R (FIG. 42A and FIG. 42B). Lack of polyspecificity was confirmed by a PSR assay (FIG. 43A-FIG. 431). AB 1084 was removed from further study due to long retention time on HIC and subsequent potential for higher aggregation propensity. Improved multispecific binding proteins demonstrated vastly higher potency than the multispecific binding protein based on the ianalumab sequence (FIG. 44A and FIG. 44B). In addition, a greater than ten-fold improvement in potency was observed as compared to the original AB0369 multispecific binding protein. Importantly, the ability to block BAFF-BAFF-R binding was significantly improved as compared to the parental AB0369 multispecific binding p...

Claims

WHAT IS CLAIMED IS:

1. A protein comprising:(a) a first antigen-binding site that binds NKG2D;(b) a second antigen-binding site that binds B cell-activating factor receptor (BAFF-R); and(c) an antibody Fc domain or a portion thereof sufficient to bind CD 16, or a third antigen-binding site that binds CD 16.

2. The protein of claim 1, wherein the first antigen-binding site that binds NKG2D is a Fab fragment, and the second antigen-binding site that binds BAFF-R is an scFv.

3. The protein of claim 1, wherein the first antigen-binding site that binds NKG2D is an scFv, and the second antigen-binding site that binds BAFF-R is a Fab fragment.

4. The protein of claim 1, further comprising an additional antigen-binding site that binds BAFF-R.

5. The protein of claim 4, wherein the first antigen-binding site that binds NKG2D is an scFv, and the second and the additional antigen-binding sites that bind BAFF-R are each a Fab fragment.

6. The protein of claim 4, wherein the first antigen-binding site that binds NKG2D is an scFv, and the second and the additional antigen-binding sites that bind BAFF-R are each an scFv.

7. The protein of any one of claims 4-6, wherein the amino acid sequences of the second and the additional antigen-binding sites are identical.

8. The protein of any one of claims 3 and 6-7, wherein the scFv that binds NKG2D is linked to an antibody constant domain or a portion thereof sufficient to bind CD 16 via a hinge comprising Ala-Ser or Gly-Ser, and wherein the scFv comprises a heavy chain variable domain and a light chain variable domain.2259. The protein of any one of claims 2 and 6-8, wherein each scFv that binds BAFF-R is linked to an antibody constant domain or a portion thereof sufficient to bind CD 16 via a hinge comprising Ala-Ser or Gly-Ser, and wherein the scFv comprises a heavy chain variable domain and a light chain variable domain.

10. The protein of claim 8 or 9, wherein the hinge further comprises an amino acid sequence Thr-Lys-Gly.

11. The protein of any one of claims 3 and 5-10, wherein within the scFv that binds NKG2D, the heavy chain variable domain of the scFv forms a disulfide bridge with the light chain variable domain of the scFv.

12. The protein of any one of claims 2 and 6-11, wherein within each scFv that binds BAFF- R, the heavy chain variable domain of the scFv forms a disulfide bridge with the light chain variable domain of the scFv.

13. The protein of claim 11 or 12, wherein the disulfide bridge is formed between C44 of the heavy chain variable domain and Cl 00 of the light chain variable domain, numbered under the Kabat numbering scheme.

14. The protein of any one of claims 3 and 5-13, wherein within the scFv that binds NKG2D, the heavy chain variable domain is linked to the light chain variable domain via a flexible linker.

15. The protein of any one of claims 2 and 6-14, wherein within each scFv that binds BAFF- R, the heavy chain variable domain is linked to the light chain variable domain via a flexible linker.

16. The protein of claim 14 or 15, wherein the flexible linker comprises (GIS)4 (SEQ ID NO: 119).

17. The protein of any one of claims 3 and 5-16, wherein within the scFv that binds NKG2D, the heavy chain variable domain is positioned at the C-terminus of the light chain variable domain.

18. The protein of any one of claims 2 and 6-17, wherein within each scFv that binds BAFF- R, the heavy chain variable domain is positioned at the C-terminus of the light chain variable domain.

19. The protein of any one of claims 3 and 5-16, wherein within the scFv that binds NKG2D, the heavy chain variable domain is positioned at the N-terminus of the light chain variable domain.

20. The protein of any one of claims 2, 6-17, and 19, wherein within each scFv that binds BAFF-R, the heavy chain variable domain is positioned at the N-terminus of the light chain variable domain.

21. The protein of any one of claims 2, 9-10, 12-13, 15-16, 18 and 20, wherein the Fab fragment that binds NKG2D is not positioned between an antigen-binding site and the Fc or the portion thereof.

22. The protein of any one of claims 3, 5, 7-8, 10-11, 13-14, 16-17, and 19, wherein no Fab fragment that binds BAFF-R is positioned between an antigen-binding site and the Fc or the portion thereof.

23. A protein comprising:(a) a first antigen-binding site comprising a Fab fragment that binds NKG2D;(b) a second antigen-binding site comprising a single-chain variable fragment (scFv) that binds B cell-activating factor receptor (BAFF-R); and(c) an Fc domain comprising a first antibody constant domain and a second antibody constant domain that form a heterodimer that binds CD 16, wherein the scFv is linked to the N-terminus of the first antibody constant domain via a hinge, and the Fab is linked to the N-terminus of the second antibody constant domain.

24. The protein of claim 23, wherein the hinge comprises Gly-Ser.

25. The protein of any one of claims 1-24, wherein the first antigen-binding site binds human NKG2D.

26. The protein of any one of claims 1-25, wherein the first antigen-binding site that binds NKG2D comprises a VH comprising complementarity-determining region 1 (CDR1), complementarity-determining region 2 (CDR2), and complementarity-determining region 3 (CDR3) comprising the amino acid sequences of SEQ ID NOs: 81, 82, and 112, respectively; and a VL comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 86, 77, and 87, respectively.

27. The protein of any one of claims 1-26, wherein the first antigen-binding site that binds NKG2D comprises a VH comprising CDR1, CDR2, and CDR3 sequences represented by the amino acid sequences of SEQ ID NOs: 81, 82, and 97, respectively; and a VL comprising CDR1, CDR2, and CDR3 sequences represented by the amino acid sequences of SEQ ID NOs: 86, 77, and 87, respectively.

28. The protein of any one of claims 1-27, wherein the first antigen-binding site that binds NKG2D comprises a VH comprising an amino acid sequence at least 90% identical to SEQ ID NO:95 and a VL comprising an amino acid sequence at least 90% identical to SEQ ID NO:85.

29. The protein of any one of claims 1-28, wherein the first antigen-binding site that binds NKG2D comprises a VH comprising an amino acid sequence of SEQ ID NO:95 and a VL comprising an amino acid sequence of SEQ ID NO:85.

30. The protein of any one of claims 1-29, wherein the second antigen-binding site comprises a heavy chain variable domain comprising CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 260, 249, and 261, respectively; and a light chain variable domain comprising CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 217, 77, and 259, respectively.

31. The protein of any one of claims 1-30, wherein the second antigen-binding site comprises a heavy chain variable domain comprising CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 214, 233, and 248, respectively; and a light chain variable domain comprising CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 217, 77, and 249, respectively.22832. The protein of claim 31, wherein the second antigen-binding site comprises a heavy chain variable domain at least 90% identical to SEQ ID NO:250 and a light chain variable domain at least 90% identical to SEQ ID NO:251.

33. The protein of claim 32, wherein the second antigen-binding site comprises a VH with a G44C substitution relative to SEQ ID NO:250, and a VL with a G100C substitution relative to SEQ ID NO:251.

34. The protein of any one of claims 1-33, wherein the second antigen-binding site comprises a VH comprising the amino acid sequence of SEQ ID NO:252 and a VL comprising the amino acid sequence of SEQ ID NO:253, or a VH comprising the amino acid sequence of SEQ ID NO:250 and a VL comprising the amino acid sequence of SEQ ID NO:251.

35. The protein of any one of claims 1-34, wherein the second antigen-binding site comprises a VH comprising the amino acid sequence of SEQ ID NO:252 and a VL comprising the amino acid sequence of SEQ ID NO: 253.

36. The protein of any one of claims 1-32 or 34, wherein the second antigen-binding site comprises a VH comprising the amino acid sequence of SEQ ID NO:250 and a VL comprising the amino acid sequence of SEQ ID NO:251.

37. The protein of any one of claims 1-35, wherein the second antigen-binding site comprises a single-chain fragment variable (scFv), and wherein the scFv comprises a VH comprising the amino acid sequence of SEQ ID NO:252 and a VL comprising the amino acid sequence of SEQ ID NO:253.

38. The protein of any one of claims 1, 2, 9-10, 12-13, 15-16, 18, 20-21, 23-35 and 37, wherein the second antigen-binding site comprises a single-chain fragment variable (scFv), and wherein the scFv comprises an amino acid sequence at least 90% identical to a sequence selected from the group consisting of SEQ ID NOs: 254 and 255.

39. The protein of any one of claims 1, 2, 9-10, 12-13, 15-16, 18, 20-21, 23-35 and 37-38, wherein the second antigen-binding site comprises an scFv and the scFv comprises an amino acid sequence at least 90% identical to SEQ ID NO:254.22940. The protein of any one of claims 1, 2, 9-10, 12-13, 15-16, 18, 20-21, 23-35 and 37-39, wherein the second antigen-binding site comprises an scFv and the scFv comprises an amino acid sequence of SEQ ID NO: 254.

41. The protein of any one of claims 1, 2, 9-10, 12-13, 15-16, 18, 20-21, 23-35 and 37-40, wherein the protein comprises an amino acid sequence at least 90% identical to SEQ ID NO:270.

42. The protein of any one of claims 1, 2, 9-10, 12-13, 15-16, 18, 20-21, 23-35 and 37-41, wherein the protein comprises an amino acid sequence of SEQ ID NO:270.

43. The protein of any one of claims 1-3, 5, 7-8, 10-11, 13-14, 16-17, 19, 24-34 and 36, wherein the protein comprises an amino acid sequence at least 90% identical to SEQ ID NO:271.

44. The protein of any one of claims 1-3, 5, 7-8, 10-11, 13-14, 16-17, 19, 24-34, 36, and 43, wherein the protein comprises the amino acid sequence of SEQ ID NO:271.

45. The protein of any one of claims 1-44, wherein the second antigen-binding site binds human BAFF-R with a dissociation constant (KD) smaller than or equal to 5 nM, as measured by surface plasmon resonance (SPR).

46. The protein of any one of claims 1-45, wherein the second antigen-binding site inhibits binding of BAFF-R to BAFF.

47. A protein comprising:(a) a first antigen-binding site comprising a VH and a VL of an anti-NKG2D antibody, wherein the VH comprises the amino acid sequence of SEQ ID NO:95 and the VL comprises the amino acid sequence of SEQ ID NO:85;(b) a second antigen-binding site comprising a VH and a VL of an anti-BAFF-R antibody, wherein the VH comprises the amino acid sequence of SEQ ID NO:252 and the VL comprises the amino acid sequence of SEQ ID NO:253; and(c) an antibody Fc domain or a portion thereof sufficient to bind CD 16, or a third antigen-binding site that binds CD 16.23048. A protein comprising:(a) a first antigen-binding site comprising a VH and a VL of an anti-NKG2D antibody, wherein the VH comprises the amino acid sequence of SEQ ID NO:95 and the VL comprises the amino acid sequence of SEQ ID NO:85;(b) a second antigen-binding site comprising the amino acid sequence of SEQ ID NO:254; and(c) an antibody Fc domain or a portion thereof sufficient to bind CD 16, or a third antigen-binding site that binds CD 16.

49. The protein of any one of claims 1-48, wherein the antibody Fc domain is a human IgGl antibody Fc domain.

50. The protein of claim 49, wherein the antibody Fc domain or a portion thereof comprises an amino acid sequence at least 90% identical to SEQ ID NO: 118.

51. The protein of claim 49 or 50, wherein at least one polypeptide chain of the antibody Fc domain comprises one or more mutations, relative to SEQ ID NO: 118, at one or more positions selected from Q347, Y349, L351, S354, E356, E357, K360, Q362, S364, T366, L368, K370, N390, K392, T394, D399, S400, D401, F405, Y407, K409, T411, and K439, numbered according to the EU numbering system.

52. The protein of any one of claims 49-51, wherein at least one polypeptide chain of the antibody Fc domain comprises one or more mutations, relative to SEQ ID NO: 118, selected from Q347E, Q347R, Y349S, Y349K, Y349T, Y349D, Y349E, Y349C, L351K, L351D, L351Y, S354C, E356K, E357Q, E357L, E357W, K360E, K360W, Q362E, S364K, S364E, S364H, S364D, T366V, T366I, T366L, T366M, T366K, T366W, T366S, L368E, L368A, L368D, K370S, N390D, N390E, K392L, K392M, K392V, K392F, K392D, K392E, T394F, D399R, D399K, D399V, S400K, S400R, D401K, F405A, F405T, F405L, Y407A, Y407I, Y407V, K409F, K409W, K409D, K409R, T41 ID, T41 IE, K439D, and K439E, numbered according to the EU numbering system.23153. The protein of any one of claims 49-52, wherein one polypeptide chain of the antibody heavy chain constant region comprises one or more mutations, relative to SEQ ID NO: 118, at one or more positions selected from Q347, Y349, L351, S354, E356, E357, K360, Q362, S364, T366, L368, K370, K392, T394, D399, S400, D401, F405, Y407, K409, T411 and K439; and the other polypeptide chain of the antibody heavy chain constant region comprises one or more mutations, relative to SEQ ID NO: 118, at one or more positions selected from Q347, Y349, L351, S354, E356, E357, S364, T366, L368, K370, N390, K392, T394, D399, D401, F405, Y407, K409, T411, and K439, numbered according to the EU numbering system.

54. The protein of claim 53, wherein one polypeptide chain of the antibody heavy chain constant region comprises K360E and K409W substitutions relative to SEQ ID NO: 118; and the other polypeptide chain of the antibody heavy chain constant region comprises Q347R, D399V and F405T substitutions relative to SEQ ID NO: 118, numbered according to the EU numbering system.

55. The protein of claim 53, wherein one polypeptide chain of the antibody heavy chain constant region comprises an F405L substitution relative to SEQ ID NO: 118; and the other polypeptide chain of the antibody heavy chain constant region comprises a K409R substitution relative to SEQ ID NO: 118, numbered according to the EU numbering system.

56. The protein of any one of claims 53-55, wherein one polypeptide chain of the antibody heavy chain constant region comprises a Y349C substitution relative to SEQ ID NO: 118; and the other polypeptide chain of the antibody heavy chain constant region comprises an S354C substitution relative to SEQ ID NO: 118, numbered according to the EU numbering system.

57. A protein comprising:(a) a first polypeptide comprising the amino acid sequence of SEQ ID NO:270;(b) a second polypeptide comprising the amino acid sequence of SEQ ID NO: 194; and(c) a third polypeptide comprising the amino acid sequence of SEQ ID NO: 195.

58. A protein comprising:232(a) a first polypeptide comprising the amino acid sequence of SEQ ID NO:271;(b) a second polypeptide comprising the amino acid sequence of SEQ ID NO:272; and(c) a third polypeptide comprising the amino acid sequence of SEQ ID NO:273.

59. A pharmaceutical composition comprising a protein according to any one of claims 1 to 58 and a pharmaceutically acceptable carrier.

60. A cell comprising one or more nucleic acids encoding a protein according to any one of claims 1 to 58.

61. A method of enhancing tumor cell death, the method comprising exposing the tumor cell and a natural killer cell to an effective amount of the protein of any one of claims 1 to 58 or the pharmaceutical composition of claim 59.

62. A method of treating cancer, the method comprising administering to a subject in need thereof an effective amount of the protein of any one of claims 1 to 58 or the pharmaceutical composition of claim 59.

63. The method according to claim 62, wherein the cancer is selected from the group consisting of B-cell non-Hodgkin’s lymphoma (B-NHL), chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), marginal zone lymphoma, mucosa-associated lymphoid tissue (MALT) lymphoma, primary mediastinal B-cell lymphoma, and acute lymphocytic leukemia (ALL).

64. A method of enhancing B cell death, the method comprising exposing the B cell and a natural killer cell to an effective amount of the protein of any one of claims 1 to 58 or the pharmaceutical composition of claim 59.

65. A method of treating an autoimmune inflammatory disease, the method comprising administering to a subject in need thereof an effective amount of the protein of any one of claims 1 to 58 or the pharmaceutical composition of claim 59.

66. A protein of any one of claims 1-58, wherein the protein is a purified protein.23367. The protein of claim 66, wherein the protein is purified using a method selected from the group consisting of: centrifugation, depth filtration, cell lysis, homogenization, freeze-thawing, affinity purification, gel filtration, ion exchange chromatography, hydrophobic interaction exchange chromatography, and mixed-mode chromatography.234

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